Electric Fields at the Active Site of an Enzyme: Direct Comparison of Experiment with Theory

NASA Astrophysics Data System (ADS)

Suydam, Ian T.; Snow, Christopher D.; Pande, Vijay S.; Boxer, Steven G.

2006-07-01

The electric fields produced in folded proteins influence nearly every aspect of protein function. We present a vibrational spectroscopy technique that measures changes in electric field at a specific site of a protein as shifts in frequency (Stark shifts) of a calibrated nitrile vibration. A nitrile-containing inhibitor is used to deliver a unique probe vibration to the active site of human aldose reductase, and the response of the nitrile stretch frequency is measured for a series of mutations in the enzyme active site. These shifts yield quantitative information on electric fields that can be directly compared with electrostatics calculations. We show that extensive molecular dynamics simulations and ensemble averaging are required to reproduce the observed changes in field.

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

PubMed Central

Sengupta, Raghuvir N.; Van Schie, Sabine N.S.; Giambaşu, George; Dai, Qing; Yesselman, Joseph D.; York, Darrin; Piccirilli, Joseph A.; Herschlag, Daniel

2016-01-01

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH2) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed MC, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. PMID:26567314

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.

PubMed

Sengupta, Raghuvir N; Van Schie, Sabine N S; Giambaşu, George; Dai, Qing; Yesselman, Joseph D; York, Darrin; Piccirilli, Joseph A; Herschlag, Daniel

2016-01-01

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. © 2015 Sengupta et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Perimeter intrusion detection and assessment system

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

Eaton, M.J.; Jacobs, J.; McGovern, D.E.

1977-11-01

To obtain an effective perimeter intrusion detection system requires careful sensor selection, procurement, and installation. The selection process involves a thorough understanding of the unique site features and how these features affect the performance of each type of sensor. It is necessary to develop procurement specifications to establish acceptable sensor performance limits. Careful explanation and inspection of critical installation dimensions is required during on-site construction. The implementation of these activities at a particular site is discussed.

Unique Kinase Catalytic Mechanism of AceK with a Single Magnesium Ion

PubMed Central

Li, Quanjie; Zheng, Jimin; Tan, Hongwei; Li, Xichen; Chen, Guangju; Jia, Zongchao

2013-01-01

Isocitrate dehydrogenase kinase/phosphatase (AceK) is the founding member of the protein phosphorylation system in prokaryotes. Based on the novel and unique structural characteristics of AceK recently uncovered, we sought to understand its kinase reaction mechanism, along with other features involved in the phosphotransfer process. Herein we report density functional theory QM calculations of the mechanism of the phosphotransfer reaction catalysed by AceK. The transition states located by the QM calculations indicate that the phosphorylation reaction, catalysed by AceK, follows a dissociative mechanism with Asp457 serving as the catalytic base to accept the proton delivered by the substrate. Our results also revealed that AceK prefers a single Mg2+-containing active site in the phosphotransfer reaction. The catalytic roles of conserved residues in the active site are discussed. PMID:23977203

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

Identification of a unique Ca2+-binding site in rat acid-sensing ion channel 3.

PubMed

Zuo, Zhicheng; Smith, Rachel N; Chen, Zhenglan; Agharkar, Amruta S; Snell, Heather D; Huang, Renqi; Liu, Jin; Gonzales, Eric B

2018-05-25

Acid-sensing ion channels (ASICs) evolved to sense changes in extracellular acidity with the divalent cation calcium (Ca 2+ ) as an allosteric modulator and channel blocker. The channel-blocking activity is most apparent in ASIC3, as removing Ca 2+ results in channel opening, with the site's location remaining unresolved. Here we show that a ring of rat ASIC3 (rASIC3) glutamates (Glu435), located above the channel gate, modulates proton sensitivity and contributes to the formation of the elusive Ca 2+ block site. Mutation of this residue to glycine, the equivalent residue in chicken ASIC1, diminished the rASIC3 Ca 2+ block effect. Atomistic molecular dynamic simulations corroborate the involvement of this acidic residue in forming a high-affinity Ca 2+ site atop the channel pore. Furthermore, the reported observations provide clarity for past controversies regarding ASIC channel gating. Our findings enhance understanding of ASIC gating mechanisms and provide structural and energetic insights into this unique calcium-binding site.

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study†

PubMed Central

Peng, Dungeng; Satterlee, James D.; Ma, Li-Hua; Dallas, Jerry L.; Smith, Kevin M.; Zhang, Xuhong; Sato, Michihiko; La Mar, Gerd N.

2011-01-01

Heme oxygenase, HO, from the pathogenic bacterium N. meningitidis, NmHO, which secures host iron, shares many properties with mammalian HOs, but also exhibits some key differences. The crystal structure appears more compact and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D 1H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~102 increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed. PMID:21870860

Develop high activity, low cost non-PGM fuel cell electrocatalyst and stable supports

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

Colon-Mercado, H. R.; Elvington, M. C.; Garcia-Diaz, B. L.

2016-09-28

A unique approach has been developed to probe the non-PGM catalyst active site for the Oxygen Reduction Reaction (ORR) for PEMFC. Iron based functionalities have been engineered into Metallic Organic Framework (MOF) catalysts to evaluate their impact on activity for the ORR. A series of FePhen@MOF catalysts have been synthesized with varying [Fe] to investigate the effect on electrochemical and electrocatalytic properties. The magnitude of the Fe II/III redox couple and the electrochemical surface area are analyzed to determine if there is a correlation between [Fe] and the ORR onset potential and/or the relative number of active sites.

Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB.

PubMed

Yu, Ta-Yi; Mok, Kenny C; Kennedy, Kristopher J; Valton, Julien; Anderson, Karen S; Walker, Graham C; Taga, Michiko E

2012-06-01

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB. Copyright © 2012 The Protein Society.

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

BECHTEL NEVADA ECOLOGICAL SERVICES

The Ecological Monitoring and Compliance program (EMAC), funded through the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO), monitors the ecosystem of the Nevada Test Site (NTS) and ensures compliance with laws and regulations pertaining to NTS biota. This report summarizes the program’s activities conducted by Bechtel Nevada (BN) during the Calendar Year 2005. Program activities included: (1) biological surveys at proposed construction sites, (2) desert tortoise compliance, (3) ecosystem mapping and data management, (4) sensitive and protected/regulated species and unique habitat monitoring, (5) habitat restoration monitoring, and (6) biological monitoring at the Non-Proliferation Test andmore » Evaluation Complex (NPTEC).« less

Combined computational and biochemical study reveals the importance of electrostatic interactions between the "pH sensor" and the cation binding site of the sodium/proton antiporter NhaA of Escherichia coli.

PubMed

Olkhova, Elena; Kozachkov, Lena; Padan, Etana; Michel, Hartmut

2009-08-15

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. The crystal structure of NhaA has provided a basis to explore the mechanism of ion exchange and its unique regulation by pH. Here, the mechanism of the pH activation of the antiporter is investigated through functional and computational studies of several variants with mutations in the ion-binding site (D163, D164). The most significant difference found computationally between the wild type antiporter and the active site variants, D163E and D164N, are low pK(a) values of Glu78 making them insensitive to pH. Although in the variant D163N the pK(a) of Glu78 is comparable to the physiological one, this variant cannot demonstrate the long-range electrostatic effect of Glu78 on the pH-dependent structural reorganization of trans-membrane helix X and, hence, is proposed to be inactive. In marked contrast, variant D164E remains sensitive to pH and can be activated by alkaline pH shift. Remarkably, as expected computationally and discovered here biochemically, D164E is viable and active in Na(+)/H(+) exchange albeit with increased apparent K(M). Our results unravel the unique electrostatic network of NhaA that connect the coupled clusters of the "pH sensor" with the binding site, which is crucial for pH activation of NhaA. 2009 Wiley-Liss, Inc.

Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C.

PubMed Central

Chauhan, Radha; Mande, Shekhar C

2002-01-01

Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism. PMID:12084012

A Phosphoenzyme Mimic, Overlapping Catalytic Sites and Reaction Coordinate Motion for Human NAMPT

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

Burgos, E.; Ho, M; Almo, S

2009-01-01

Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD+) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF3- as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reactionmore » coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-N1 while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.« less

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

PubMed Central

Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

2016-01-01

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

Salicylic Acid and Jasmonic Acid Pathways are Activated in Spatially Different Domains Around the Infection Site During Effector-Triggered Immunity in Arabidopsis thaliana.

PubMed

Betsuyaku, Shigeyuki; Katou, Shinpei; Takebayashi, Yumiko; Sakakibara, Hitoshi; Nomura, Nobuhiko; Fukuda, Hiroo

2018-01-01

The innate immune response is, in the first place, elicited at the site of infection. Thus, the host response can be different among the infected cells and the cells surrounding them. Effector-triggered immunity (ETI), a form of innate immunity in plants, is triggered by specific recognition between pathogen effectors and their corresponding plant cytosolic immune receptors, resulting in rapid localized cell death known as hypersensitive response (HR). HR cell death is usually limited to a few cells at the infection site, and is surrounded by a few layers of cells massively expressing defense genes such as Pathogenesis-Related Gene 1 (PR1). This virtually concentric pattern of the cellular responses in ETI is proposed to be regulated by a concentration gradient of salicylic acid (SA), a phytohormone accumulated around the infection site. Recent studies demonstrated that jasmonic acid (JA), another phytohormone known to be mutually antagonistic to SA in many cases, is also accumulated in and required for ETI, suggesting that ETI is a unique case. However, the molecular basis for this uniqueness remained largely to be solved. Here, we found that, using intravital time-lapse imaging, the JA signaling pathway is activated in the cells surrounding the central SA-active cells around the infection sites in Arabidopsis thaliana. This distinct spatial organization explains how these two phythormone pathways in a mutually antagonistic relationship can be activated simultaneously during ETI. Our results re-emphasize that the spatial consideration is a key strategy to gain mechanistic insights into the apparently complex signaling cross-talk in immunity. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase.

PubMed

Smith, Andrew T; Doyle, Wendy A; Dorlet, Pierre; Ivancich, Anabella

2009-09-22

The surface oxidation site (Trp-171) in lignin peroxidase (LiP) required for the reaction with veratryl alcohol a high-redox-potential (1.4 V) substrate, was engineered into Coprinus cinereus peroxidase (CiP) by introducing a Trp residue into a heme peroxidase that has similar protein fold but lacks this activity. To create the catalytic activity toward veratryl alcohol in CiP, it was necessary to reproduce the Trp site and its negatively charged microenvironment by means of a triple mutation. The resulting D179W+R258E+R272D variant was characterized by multifrequency EPR spectroscopy. The spectra unequivocally showed that a new Trp radical [g values of g(x) = 2.0035(5), g(y) = 2.0027(5), and g(z) = 2.0022(1)] was formed after the [Fe(IV)=O Por(*+)] intermediate, as a result of intramolecular electron transfer between Trp-179 and the porphyrin. Also, the EPR characterization crucially showed that [Fe(IV)=O Trp-179(*)] was the reactive intermediate with veratryl alcohol. Accordingly, our work shows that it is necessary to take into account the physicochemical properties of the radical, fine-tuned by the microenvironment, as well as those of the preceding [Fe(IV)=O Por(*+)] intermediate to engineer a catalytically competent Trp site for a given substrate. Manipulation of the microenvironment of the Trp-171 site in LiP allowed the detection by EPR spectroscopy of the Trp-171(*), for which direct evidence has been missing so far. Our work also highlights the role of Trp residues as tunable redox-active cofactors for enzyme catalysis in the context of peroxidases with a unique reactivity toward recalcitrant substrates that require oxidation potentials not realized at the heme site.

Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

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

Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

2014-04-15

In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies.more » The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.« less

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

PubMed Central

Jagtap, Pravin Kumar Ankush; Soni, Vijay; Vithani, Neha; Jhingan, Gagan Deep; Bais, Vaibhav Singh; Nandicoori, Vinay Kumar; Prakash, Balaji

2012-01-01

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmUMtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmUMtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmUMtb. In this SN2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmUMtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmUMtb, which may be exploited for the development of inhibitors specific to GlmU. PMID:22969087

Chapter 19: Catalysis by Metal Carbides and Nitrides

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

Schaidle, Joshua A; Nash, Connor P; Yung, Matthew M

Early transition metal carbides and nitrides (ETMCNs), materials in which carbon or nitrogen occupies interstitial sites within a parent metal lattice, possess unique physical and chemical properties that motivate their use as catalysts. Specifically, these materials possess multiple types of catalytic sites, including metallic, acidic, and basic sites, and as such, exhibit reactivities that differ from their parent metals. Moreover, their surfaces are dynamic under reaction conditions. This chapter reviews recent (since 2010) experimental and computational investigations into the catalytic properties of ETMCN materials for applications including biomass conversion, syngas and CO2 upgrading, petroleum and natural gas refining, and electrocatalyticmore » energy conversion, energy storage, and chemicals production, and attempts to link catalyst performance to active site identity/surface structure in order to elucidate the present level of understanding of structure-function relationships for these materials. The chapter concludes with a perspective on leveraging the unique properties of these materials to design and develop improved catalysts through a dedicated, multidisciplinary effort.« less

Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism*

PubMed Central

Murray, David S.; Chinnam, Nagababu; Tonthat, Nam Ky; Whitfill, Travis; Wray, Lewis V.; Fisher, Susan H.; Schumacher, Maria A.

2013-01-01

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg62, from an adjacent subunit. Notably, Arg62 must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens. PMID:24158439

Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry.

PubMed

Swaney, Danielle L; Wenger, Craig D; Thomson, James A; Coon, Joshua J

2009-01-27

Protein phosphorylation is central to the understanding of cellular signaling, and cellular signaling is suggested to play a major role in the regulation of human embryonic stem (ES) cell pluripotency. Here, we describe the use of conventional tandem mass spectrometry-based sequencing technology--collision-activated dissociation (CAD)--and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome. In total, these experiments resulted in the identification of 11,995 unique phosphopeptides, corresponding to 10,844 nonredundant phosphorylation sites, at a 1% false discovery rate (FDR). Among these phosphorylation sites are 5 localized to 2 pluripotency critical transcription factors--OCT4 and SOX2. From these experiments, we conclude that ETD identifies a larger number of unique phosphopeptides than CAD (8,087 to 3,868), more frequently localizes the phosphorylation site to a specific residue (49.8% compared with 29.6%), and sequences whole classes of phosphopeptides previously unobserved.

The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis

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

Van Bueren, A.; Ghinet, M; Gregg, K

2009-01-01

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with ?-galactosidase activity (Escherichia coli LacZ), ?-glucuronidase activity (Homo sapiens GusB), and ?-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-?-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role ofmore » E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural ?-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-?-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.« less

The Structural Basis of Substrate Recognition in an exo-beta-d-Glucosaminidase Involved in Chitosan Hydrolysis

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

Lammerts van Bueren, A.; Ghinet, M; Gregg, K

2009-01-01

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with beta-galactosidase activity (Escherichia coli LacZ), beta-glucuronidase activity (Homo sapiens GusB), and beta-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-beta-D-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role ofmore » E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural beta-1,4-D-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-beta-D-glucosaminide synthetic substrate provide insight into interactions in the +1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.« less

A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase.

PubMed

Goris, Tobias; Wait, Annemarie F; Saggu, Miguel; Fritsch, Johannes; Heidary, Nina; Stein, Matthias; Zebger, Ingo; Lendzian, Friedhelm; Armstrong, Fraser A; Friedrich, Bärbel; Lenz, Oliver

2011-05-01

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.

Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein–protein interactions

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

Kumar, Shiva; Krishnamoorthy, Kalyanaraman; Mudeppa, Devaraja G.

P. falciparum orotate phosphoribosyltransferase, a potential target for antimalarial drugs and a conduit for prodrugs, crystallized as a structure with eight molecules per asymmetric unit that included some unique parasite-specific auto-inhibitory interactions between catalytic dimers. The most severe form of malaria is caused by the obligate parasite Plasmodium falciparum. Orotate phosphoribosyltransferase (OPRTase) is the fifth enzyme in the de novo pyrimidine-synthesis pathway in the parasite, which lacks salvage pathways. Among all of the malaria de novo pyrimidine-biosynthesis enzymes, the structure of P. falciparum OPRTase (PfOPRTase) was the only one unavailable until now. PfOPRTase that could be crystallized was obtained aftermore » some low-complexity sequences were removed. Four catalytic dimers were seen in the asymmetic unit (a total of eight polypeptides). In addition to revealing unique amino acids in the PfOPRTase active sites, asymmetric dimers in the larger structure pointed to novel parasite-specific protein–protein interactions that occlude the catalytic active sites. The latter could potentially modulate PfOPRTase activity in parasites and possibly provide new insights for blocking PfOPRTase functions.« less

Differential Sox10 Genomic Occupancy in Myelinating Glia

PubMed Central

Lopez-Anido, Camila; Sun, Guannan; Koenning, Matthias; Srinivasan, Rajini; Hung, Holly A.; Emery, Ben; Keles, Sunduz; Svaren, John

2015-01-01

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells, and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. PMID:25974668

In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed.

PubMed

Ochsner, Andrea M; Müller, Jonas E N; Mora, Carlos A; Vorholt, Julia A

2014-08-25

In the Gram-positive methylotroph Bacillus methanolicus, methanol oxidation is catalyzed by an NAD-dependent methanol dehydrogenase (Mdh) that belongs to the type III alcohol dehydrogenase (Adh) family. It was previously shown that the in vitro activity of B. methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase. Here we show that this feature is not unique, but more widespread among type III Adhs in combination with Act or other Act-like Nudix hydrolases. In addition, we studied the effect of site directed mutations in the predicted active site of Mdh and two other type III Adhs with regard to activity and activation by Act. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of themore » domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.« less

Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold.

PubMed

Feliciano, Patricia R; Drennan, Catherine L; Nonato, M Cristina

2016-08-30

Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases.

Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by 15N and 2H ESEEM Spectroscopy

PubMed Central

Vogt, Matthew; Lahiri, Simanti; Hoogstraten, Charles G.; Britt, R. David; DeRose, Victoria J.

2010-01-01

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a Kd,app < 10 µM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin–echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands. PMID:17177426

A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer

PubMed Central

Good, Charly R.; Madzo, Jozef; Patel, Bela; Maegawa, Shinji; Engel, Nora; Jelinek, Jaroslav

2017-01-01

Abstract TET1 oxidizes methylated cytosine into 5-hydroxymethylcytosine (5hmC), resulting in regulation of DNA methylation and gene expression. Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated CpG islands (CGIs). This CXXC domain allows TET1 to protect CGIs from aberrant methylation, but it also limits its ability to regulate genes outside of CGIs. Here, we report a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site. Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is repressed in embryonic stem cells (ESCs) but becomes activated in embryonic and adult tissues while TET1FL is expressed in ESCs, but repressed in adult tissues. Overexpression of TET1ALT shows production of 5hmC with distinct (and weaker) effects on DNA methylation or gene expression when compared to TET1FL. TET1ALT is aberrantly activated in multiple cancer types including breast, uterine and glioblastoma, and TET1 activation is associated with a worse overall survival in breast, uterine and ovarian cancers. Our data suggest that the predominantly activated isoform of TET1 in cancer cells does not protect from CGI methylation and likely mediates dynamic site-specific demethylation outside of CGIs. PMID:28531272

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

PubMed Central

Kirouac, Kevin N.; Ling, Hong

2011-01-01

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι’s biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O8 atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase. PMID:21300901

Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone

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

Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan

Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A activemore » site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.« less

External Barium Affects the Gating of KCNQ1 Potassium Channels and Produces a Pore Block via Two Discrete Sites

PubMed Central

Gibor, Gilad; Yakubovich, Daniel; Peretz, Asher; Attali, Bernard

2004-01-01

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba2+ binding kinetics and the concentration and voltage dependence of Ba2+ steady-state block. Our results indicate that extracellular Ba2+ exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba2+ site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba2+ site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba2+ on channel gating in low external K+ solutions. Ba2+ binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K+ attenuates Ba2+ inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K+ channels, KCNQ1 channels display significant structural and functional uniqueness. PMID:15226366

Structural Mechanisms of Plant Glucan Phosphatases in Starch Metabolism

PubMed Central

Meekins, David A.; Vander Kooi, Craig W.; Gentry, Matthew S.

2016-01-01

Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode for two glucan phosphatases called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2) that regulate starch metabolism by selectively dephosphorylating glucose moieties within starch glucan chains. Recently, the structures of both SEX4 and LSF2 were determined, with and without phosphoglucan products bound, revealing the mechanism for their unique activities. This review explores the structural and enzymatic features of the plant glucan phosphatases and outlines how they are uniquely adapted for carrying out their cellular functions. We outline the physical mechanisms employed by SEX4 and LSF2 to interact with starch glucans: SEX4 binds glucan chains via a continuous glucan binding platform comprised of its Dual Specificity Phosphatase (DSP) domain and Carbohydrate Binding Module (CBM) while LSF2 utilizes Surface Binding Sites (SBSs). SEX4 and LSF2 both contain a unique network of aromatic residues in their catalytic DSP domains that serve as glucan engagement platforms and are unique to the glucan phosphatases. We also discuss the phosphoglucan substrate specificities inherent to SEX4 and LSF2 and outline structural features within the active site that govern glucan orientation. This review defines the structural mechanism of the plant glucan phosphatases with respect to phosphatases, starch metabolism, and protein-glucan interaction; thereby providing a framework for their applications in both agricultural and industrial settings. PMID:26934589

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

PubMed

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

2014-03-01

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

CDC/NACCHO Accreditation Support Initiative: advancing readiness for local and tribal health department accreditation.

PubMed

Monteiro, Erinn; Fisher, Jessica Solomon; Daub, Teresa; Zamperetti, Michelle Chuk

2014-01-01

Health departments have various unique needs that must be addressed in preparing for national accreditation. These needs require time and resources, shortages that many health departments face. The Accreditation Support Initiative's goal was to test the assumption that even small amounts of dedicated funding can help health departments make important progress in their readiness to apply for and achieve accreditation. Participating sites' scopes of work were unique to the needs of each site and based on the proposed activities outlined in their applications. Deliverables and various sources of data were collected from sites throughout the project period (December 2011-May 2012). Awardees included 1 tribal and 12 local health departments, as well as 5 organizations supporting the readiness of local and tribal health departments. Sites dedicated their funding toward staff time, accreditation fees, completion of documentation, and other accreditation readiness needs and produced a number of deliverables and example documents. All sites indicated that they made accreditation readiness gains that would not have occurred without this funding. Preliminary evaluation data from the first year of the Accreditation Support Initiative indicate that flexible funding arrangements may be an effective way to increase health departments' accreditation readiness.

Functions and Unique Diversity of Genes and Microorganisms Involved in Arsenite Oxidation from the Tailings of a Realgar Mine

PubMed Central

E, Guoji; Wang, Jianing; Wang, Nian; Chen, Xiaoming; Mu, Yao; Li, Hao; Yang, Ye; Liu, Yichen; Wang, Yanxin

2016-01-01

ABSTRACT The tailings of the Shimen realgar mine have unique geochemical features. Arsenite oxidation is one of the major biogeochemical processes that occurs in the tailings. However, little is known about the functional and molecular aspects of the microbial community involved in arsenite oxidation. Here, we fully explored the functional and molecular features of the microbial communities from the tailings of the Shimen realgar mine. We collected six samples of tailings from sites A, B, C, D, E, and F. Microcosm assays indicated that all of the six sites contain both chemoautotrophic and heterotrophic arsenite-oxidizing microorganisms; their activities differed considerably from each other. The microbial arsenite-oxidizing activities show a positive correlation with soluble arsenic concentrations. The microbial communities of the six sites contain 40 phyla of bacteria and 2 phyla of archaea that show extremely high diversity. Soluble arsenic, sulfate, pH, and total organic carbon (TOC) are the key environmental factors that shape the microbial communities. We further identified 114 unique arsenite oxidase genes from the samples; all of them code for new or new-type arsenite oxidases. We also isolated 10 novel arsenite oxidizers from the samples, of which 4 are chemoautotrophic and 6 are heterotrophic. These data highlight the unique diversities of the arsenite-oxidizing microorganisms and their oxidase genes from the tailings of the Shimen realgar mine. To the best of our knowledge, this is the first report describing the functional and molecular features of microbial communities from the tailings of a realgar mine. IMPORTANCE This study focused on the functional and molecular characterizations of microbial communities from the tailings of the Shimen realgar mine. We fully explored, for the first time, the arsenite-oxidizing activities and the functional gene diversities of microorganisms from the tailings, as well as the correlation of the microbial activities/diversities with environmental factors. The findings of this study help us to better understand the diversities of the arsenite-oxidizing bacteria and the geochemical cycle of arsenic in the tailings of the Shimen realgar mine and gain insights into the microbial mechanisms by which the secondary minerals of the tailings were formed. This work also offers a set of unique arsenite-oxidizing bacteria for basic research of the molecular regulation of arsenite oxidation in bacterial cells and for the environmentally friendly bioremediation of arsenic-contaminated groundwater. PMID:27663031

Functions and Unique Diversity of Genes and Microorganisms Involved in Arsenite Oxidation from the Tailings of a Realgar Mine.

PubMed

Zeng, Xian-Chun; E, Guoji; Wang, Jianing; Wang, Nian; Chen, Xiaoming; Mu, Yao; Li, Hao; Yang, Ye; Liu, Yichen; Wang, Yanxin

2016-12-15

The tailings of the Shimen realgar mine have unique geochemical features. Arsenite oxidation is one of the major biogeochemical processes that occurs in the tailings. However, little is known about the functional and molecular aspects of the microbial community involved in arsenite oxidation. Here, we fully explored the functional and molecular features of the microbial communities from the tailings of the Shimen realgar mine. We collected six samples of tailings from sites A, B, C, D, E, and F. Microcosm assays indicated that all of the six sites contain both chemoautotrophic and heterotrophic arsenite-oxidizing microorganisms; their activities differed considerably from each other. The microbial arsenite-oxidizing activities show a positive correlation with soluble arsenic concentrations. The microbial communities of the six sites contain 40 phyla of bacteria and 2 phyla of archaea that show extremely high diversity. Soluble arsenic, sulfate, pH, and total organic carbon (TOC) are the key environmental factors that shape the microbial communities. We further identified 114 unique arsenite oxidase genes from the samples; all of them code for new or new-type arsenite oxidases. We also isolated 10 novel arsenite oxidizers from the samples, of which 4 are chemoautotrophic and 6 are heterotrophic. These data highlight the unique diversities of the arsenite-oxidizing microorganisms and their oxidase genes from the tailings of the Shimen realgar mine. To the best of our knowledge, this is the first report describing the functional and molecular features of microbial communities from the tailings of a realgar mine. This study focused on the functional and molecular characterizations of microbial communities from the tailings of the Shimen realgar mine. We fully explored, for the first time, the arsenite-oxidizing activities and the functional gene diversities of microorganisms from the tailings, as well as the correlation of the microbial activities/diversities with environmental factors. The findings of this study help us to better understand the diversities of the arsenite-oxidizing bacteria and the geochemical cycle of arsenic in the tailings of the Shimen realgar mine and gain insights into the microbial mechanisms by which the secondary minerals of the tailings were formed. This work also offers a set of unique arsenite-oxidizing bacteria for basic research of the molecular regulation of arsenite oxidation in bacterial cells and for the environmentally friendly bioremediation of arsenic-contaminated groundwater. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

X-ray Crystallographic Analysis of [alpha]-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

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

Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine

2010-11-03

Three cocrystal X-ray structures of the {alpha}-ketoheterocycle inhibitors 3-5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the {alpha}-ketoheterocycle inhibitors captured as deprotonated hemiketals mimickingmore » the tetrahedral intermediate of the enzyme-catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure-activity relationships are discussed providing important insights for future design.« less

Nano-organocatalyst: Magnetically retrievable ferrite-anchored glutathione for microwave-assisted Paal-Knorr reaction, Aza-Michael addition and pyrazole synthesis

EPA Science Inventory

Postsynthetic surface modification of magnetic nanoparticles by glutathione imparts desirable chemical functionality and enables the generation of catalytic sites on the surfaces of ensuing organocatalysts. In this article, we discuss the developments, unique activity and high s...

Primary structural variation in anaplasma marginale Msp2 efficiently generates immune escape variants

USDA-ARS?s Scientific Manuscript database

Antigenic variation allows microbial pathogens to evade immune clearance and establish persistent infection. Anaplasma marginale utilizes gene conversion of a repertoire of silent msp2 alleles into a single active expression site to encode unique Msp2 variants. As the genomic complement of msp2 alle...

78 FR 56720 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-13

...-valve replacement in high-risk patients. N Engl J Med. 2011 Jun 9;364(23):2187-98. [PMID 21639811... be activated upon demand to release the therapeutic agent at the desired site. The concurrent release... streamlined for high-throughput analysis. Quantitative molecular diagnostics. Unique microRNAs and/or mRNAs...

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

PubMed Central

Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

2015-01-01

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution. PMID:26673816

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

NASA Astrophysics Data System (ADS)

Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

2015-12-01

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry

PubMed Central

Swaney, Danielle L.; Wenger, Craig D.; Thomson, James A.; Coon, Joshua J.

2009-01-01

Protein phosphorylation is central to the understanding of cellular signaling, and cellular signaling is suggested to play a major role in the regulation of human embryonic stem (ES) cell pluripotency. Here, we describe the use of conventional tandem mass spectrometry-based sequencing technology—collision-activated dissociation (CAD)—and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome. In total, these experiments resulted in the identification of 11,995 unique phosphopeptides, corresponding to 10,844 nonredundant phosphorylation sites, at a 1% false discovery rate (FDR). Among these phosphorylation sites are 5 localized to 2 pluripotency critical transcription factors—OCT4 and SOX2. From these experiments, we conclude that ETD identifies a larger number of unique phosphopeptides than CAD (8,087 to 3,868), more frequently localizes the phosphorylation site to a specific residue (49.8% compared with 29.6%), and sequences whole classes of phosphopeptides previously unobserved. PMID:19144917

The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

PubMed Central

Just, Victoria J.; Burrell, Matthew R.; Bowater, Laura; McRobbie, Iain; Stevenson, Clare E. M.; Lawson, David M.; Bornemann, Stephen

2007-01-01

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural. PMID:17680775

An alternate binding site for PPARγ ligands

PubMed Central

Hughes, Travis S.; Giri, Pankaj Kumar; de Vera, Ian Mitchelle S.; Marciano, David P.; Kuruvilla, Dana S.; Shin, Youseung; Blayo, Anne-Laure; Kamenecka, Theodore M.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

2014-01-01

PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands. PMID:24705063

Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides

NASA Astrophysics Data System (ADS)

Crittenden, Christopher M.; Parker, W. Ryan; Jenner, Zachary B.; Bruns, Kerry A.; Akin, Lucas D.; McGee, William M.; Ciccimaro, Eugene; Brodbelt, Jennifer S.

2016-05-01

A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.

Cloning and expression in Escherichia coli of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

PubMed

Zalacain, M; Malpartida, F; Pulido, D; Jiménez, A

1987-01-15

The Streptomyces hygroscopicus hyg gene encoding a hygromycin B phosphotransferase has been introduced into different sites of both the Escherichia coli plasmid pBR322 and the Escherichia coli-Saccharomyces cerevisiae shuttle vector YRp7. When this gene was inserted into the BamHI site of pBR322 and then cloned in E. coli phosphorylating activity was not detected, indicating that the hyg gene promoter was not functional in this bacterium. However, when the hyg gene was inserted into either the unique PstI site of pBR322 or into each of the two PstI sites of YRp7, phosphotransferase activity was observed. Analysis of the translation products from these constructions by coupled in vitro transcription-translation systems suggested that in all cases transcrition was regulated by a promoter not provided by the inserted hyg gene and that the synthesized polypeptide was identical to that present in S. hygroscopicus.

Amino Acid Residues That Contribute to Substrate Specificity of Class A β-Lactamase SME-1

PubMed Central

Majiduddin, Fahd K.; Palzkill, Timothy

2005-01-01

Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by β-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 β-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A β-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of β-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket. PMID:16048956

Amino acid residues that contribute to substrate specificity of class A beta-lactamase SME-1.

PubMed

Majiduddin, Fahd K; Palzkill, Timothy

2005-08-01

Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by beta-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 beta-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A beta-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of beta-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

The EarthScope Transportable Array Migrates Eastward: Engaging the Science Community and Students

NASA Astrophysics Data System (ADS)

Dorr, P. M.; Busby, R. W.; Hafner, K.; Taber, J.; Woodward, R.

2009-12-01

The EarthScope Transportable Array (TA) is at the midway point of its ten-year migration from the Pacific to the Atlantic coasts of North America. In 2010, TA activities will begin on the eastern side of the Mississippi River, and will be fully deployed around the New Madrid region for the 2011-2012 bicentennial of these historic earthquakes. As the TA migrates eastward, it supports outreach activities to increase awareness and understanding of seismology concepts and scientific discoveries enabled by the EarthScope facilities, including several in collaboration with the EarthScope National Office and the Plate Boundary Observatory. The TA also has a goal of actively engaging students who will become the next generation of Earth scientists. The TA contributes to this goal by offering university students an opportunity to perform site reconnaissance for future seismic stations. Through its Student Siting Program, the TA provides a unique opportunity for scientists and students to become directly involved in the TA. From 2005 to 2009, about 90 students from 31 universities conducted site reconnaissance for more than 835 sites across the western half of the US. The students are supervised by faculty drawn from a number of universities in the siting region, thus further increasing the involvement in USArray. In the summer of 2010, participants in the Student Siting Program will identify sites in Michigan's Upper Peninsula, Wisconsin, Illinois, western Kentucky, western Tennessee, Mississippi and Alabama. Universities, regional seismic networks, and other interested organizations have the unique opportunity to adopt one or more installed, fully operational Transportable Array stations at the end of their two-year deployments. Such adopted stations become a permanent resource for educational and research seismology. In addition, EarthScope and USArray provide a range of outreach materials that support geoscientists in their own regional outreach efforts. For example, the EarthScope onSite newsletter and other publications can be used for outreach to colleagues, schools, and the general public to communicate the excitement and scientific discoveries of EarthScope. Other outreach activities include teacher workshops, classroom seismographs and a DVD of earthquake-related educational materials, and EarthScope-specific and regional-specific pages for the Active Earth interactive display. We will present TA deployment maps and schedules, comprehensive information about the station adoption and siting reconnaissance programs, and examples of outreach materials to facilitate and support the science community’s involvement in EarthScope as it moves into the continental interior.

Unique battery with an active membrane separator having uniform physico-chemically functionalized ion channels and a method making the same

DOEpatents

Gerald, II, Rex E.; Ruscic, Katarina J [Chicago, IL; Sears, Devin N [Spruce Grove, CA; Smith, Luis J [Natick, MA; Klingler, Robert J [Glenview, IL; Rathke, Jerome W [Homer Glen, IL

2012-02-21

The invention relates to a unique battery having an active, porous membrane and method of making the same. More specifically the invention relates to a sealed battery system having a porous, metal oxide membrane with uniform, physicochemically functionalized ion channels capable of adjustable ionic interaction. The physicochemically-active porous membrane purports dual functions: an electronic insulator (separator) and a unidirectional ion-transporter (electrolyte). The electrochemical cell membrane is activated for the transport of ions by contiguous ion coordination sites on the interior two-dimensional surfaces of the trans-membrane unidirectional pores. The membrane material is designed to have physicochemical interaction with ions. Control of the extent of the interactions between the ions and the interior pore walls of the membrane and other materials, chemicals, or structures contained within the pores provides adjustability of the ionic conductivity of the membrane.

Structural and functional determination of homologs of the Mycobacterium tuberculosisN-acetylglucosamine-6-phosphate deacetylase (NagA).

PubMed

Ahangar, Mohd Syed; Furze, Christopher M; Guy, Collette S; Cooper, Charlotte; Maskew, Kathryn S; Graham, Ben; Cameron, Alexander D; Fullam, Elizabeth

2018-05-04

The Mycobacterium tuberculosis (Mtb) pathogen encodes an N -acetylglucosamine-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of N -acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential anti-tubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereo-selective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 Å and 2.0 Å resolutions, respectively. The GlcNAc6P-complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Structures of minimal catalytic fragments of topoisomerase V reveals conformational changes relevant for DNA binding

PubMed Central

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

2010-01-01

Summary Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix hairpin helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and shows how topoisomerase V may interact with DNA. PMID:20637419

Kinetic Contributions to Gating by Interactions Unique to N-methyl-d-aspartate (NMDA) Receptors*

PubMed Central

Borschel, William F.; Cummings, Kirstie A.; Tindell, LeeAnn K.; Popescu, Gabriela K.

2015-01-01

Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses. PMID:26370091

Cloning and characterization of microbial activated Aedes aegypti MEK4 (AaMEK4): influences of noncatalytic domains on enzymatic activity.

PubMed

Wu, R C-C; Cho, W-L

2014-10-01

Protein kinases are known to be involved in a number of signal transduction cascades. Both the stress-activated Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) p38 pathways have been shown to correlate with the insect immune response to microbial infection. MAP kinase kinase 4 (MEK4) is an upstream kinase of JNK and p38 kinase. The cDNA of AaMEK4 was cloned and characterized. AaMEK4 was activated by microbial lysates of Gram-positive, Gram-negative bacteria and yeast. The conserved lysine (K112 ) and the putative phosphorylation sites (S238 and T242 ) were shown to be important for kinase activity by site-directed mutagenesis. A common MAPK docking site (MAPK_dsA) was found and in addition, a new nearby docking site, MAPK_dsB, was identified in the N-terminal noncatalytic domain of AaMEK4. MAPK_dsB was shown to be a unique element in the MEK4 family. In this study, both MAPK_dsA and _dsB were demonstrated to be important to AaMEK4 enzymatic activity for the downstream protein kinase, Aap38. © 2014 The Royal Entomological Society.

The impact of social media on children, adolescents, and families.

PubMed

O'Keeffe, Gwenn Schurgin; Clarke-Pearson, Kathleen

2011-04-01

Using social media Web sites is among the most common activity of today's children and adolescents. Any Web site that allows social interaction is considered a social media site, including social networking sites such as Facebook, MySpace, and Twitter; gaming sites and virtual worlds such as Club Penguin, Second Life, and the Sims; video sites such as YouTube; and blogs. Such sites offer today's youth a portal for entertainment and communication and have grown exponentially in recent years. For this reason, it is important that parents become aware of the nature of social media sites, given that not all of them are healthy environments for children and adolescents. Pediatricians are in a unique position to help families understand these sites and to encourage healthy use and urge parents to monitor for potential problems with cyberbullying, "Facebook depression," sexting, and exposure to inappropriate content.

Ecological Monitoring and Compliance Program Fiscal/Calendar Year 2004 Report

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

Bechtel Nevada

2005-03-01

The Ecological Monitoring and Compliance program, funded through the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office, monitors the ecosystem of the Nevada Test Site and ensures compliance with laws and regulations pertaining to Nevada Test Site biota. This report summarizes the program's activities conducted by Bechtel Nevada during the Fiscal Year 2004 and the additional months of October, November, and December 2004, reflecting a change in the monitoring period to a calendar year rather than a fiscal year as reported in the past. This change in the monitoring period was made to better accommodate information requiredmore » for the Nevada Test Site Environmental Report, which reports on a calendar year rather than a fiscal year. Program activities included: (1) biological surveys at proposed construction sites, (2) desert tortoise compliance, (3) ecosystem mapping and data management, (4) sensitive species and unique habitat monitoring, (5) habitat restoration monitoring, and (6) biological monitoring at the Hazardous Materials Spill Center.« less

Support effects in catalysis studied by in-situ sum frequency generation vibrational spectroscopy and in-situ x-ray spectroscopies

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

Kennedy, Griffin John

Here, kinetic measurements are paired with in-situ spectroscopic characterization tools to investigate colloidally based, supported Pt catalytic model systems in order to elucidate the mechanisms by which metal and support work in tandem to dictate activity and selectivity. The results demonstrate oxide support materials, while inactive in absence of Pt nanoparticles, possess unique active sites for the selective conversion of gas phase molecules when paired with an active metal catalyst.

The Enzymatic and Structural Basis for Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I).

PubMed

Salinas, Gustavo; Gao, Wei; Wang, Yang; Bonilla, Mariana; Yu, Long; Novikov, Andrey; Virginio, Veridiana G; Ferreira, Henrique B; Vieites, Marisol; Gladyshev, Vadim N; Gambino, Dinorah; Dai, Shaodong

2017-12-20

New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with Au I -MPO, a novel gold inhibitor, together with inhibition assays were performed. Au I -MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer-monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxin (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys 519 and Cys 573 in the Au I -TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys 519 and Cys 573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491-1504.

The Enzymatic and Structural Basis for Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I)

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

Salinas, Gustavo; Gao, Wei; Wang, Yang

Aims: New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with AuI-MPO, a novel gold inhibitor, together with inhibition assays were performed. Results: AuI-MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer–monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxinmore » (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys519 and Cys573 in the AuI-TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. Innovation: The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. Conclusions: The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys519 and Cys573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491–1504.« less

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site.

PubMed

Sayer, Christopher; Finnigan, William; Isupov, Michail N; Levisson, Mark; Kengen, Servé W M; van der Oost, John; Harmer, Nicholas J; Littlechild, Jennifer A

2016-05-10

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

PubMed Central

Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

2016-01-01

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

Participatory role of zinc in structural and functional characterization of bioremediase: a unique thermostable microbial silica leaching protein.

PubMed

Chowdhury, Trinath; Sarkar, Manas; Chaudhuri, Biswadeep; Chattopadhyay, Brajadulal; Halder, Umesh Chandra

2015-07-01

A unique protein, bioremediase (UniProt Knowledgebase Accession No.: P86277), isolated from a hot spring bacterium BKH1 (GenBank Accession No.: FJ177512), has shown to exhibit silica leaching activity when incorporated to prepare bio-concrete material. Matrix-assisted laser desorption ionization mass spectrometry analysis suggests that bioremediase is 78% homologous to bovine carbonic anhydrase II though it does not exhibit carbonic anhydrase-like activity. Bioinformatics study is performed for understanding the various physical and chemical parameters of the protein which predicts the involvement of zinc encircled by three histidine residues (His94, His96 and His119) at the active site of the protein. Isothermal titration calorimetric-based thermodynamic study on diethyl pyrocarbonate-modified protein recognizes the presence of Zn(2+) in the enzyme moiety. Exothermic to endothermic transition as observed during titration of the protein with Zn(2+) discloses that there are at least two binding sites for zinc within the protein moiety. Addition of Zn(2+) regains the activity of EDTA chelated bioremediase confirming the presence of extra binding site of Zn(2+) in the protein moiety. Revival of folding pattern of completely unfolded urea-treated protein by Zn(2+) explains the participatory role of zinc in structural stability of the protein. Restoration of the λ max in intrinsic fluorescence emission study of the urea-treated protein by Zn(2+) similarly confirms the involvement of Zn in the refolding of the protein. The utility of bioremediase for silica nanoparticles preparation is observed by field emission scanning electron microscopy.

SUMMARY OF TECNIQUES AND UNIQUE USES FOR DIRECT PUSH METHODS IN SITE CHARACTERIZATION ON CONTAMINATED FIELD SITES

EPA Science Inventory

At many of the sites where we have been asked to assist in site characterization, we have discovered severe discrepancies that new technologies may be able to prevent. This presentation is designed to illustrate these new technologies or unique uses of existing technology and the...

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

PubMed

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

2016-08-01

Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase▿ †

PubMed Central

tenBrink, Felix; Schink, Bernhard; Kroneck, Peter M. H.

2011-01-01

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H2O to acetylene (H—C☰C—H) to form acetaldehyde (CH3CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme. PMID:21193613

The Planned and the Emergent: An Alternative Model of Learning and Literacy

ERIC Educational Resources Information Center

Rogers, Lori S.

2013-01-01

Within academic institutions, writing centers are uniquely situated, socially rich sites for exploring learning and literacy. I examine the work of the Michigan Tech Writing Center's UN 1002 World Cultures study teams primarily because student participants and Writing Center coaches are actively engaged in structuring their own learning and…

A survey of health-related activities on second life.

PubMed

Beard, Leslie; Wilson, Kumanan; Morra, Dante; Keelan, Jennifer

2009-05-22

Increasingly, governments, health care agencies, companies, and private groups have chosen Second Life as part of their Web 2.0 communication strategies. Second Life offers unique design features for disseminating health information, training health professionals, and enabling patient education for both academic and commercial health behavior research. This study aimed to survey and categorize the range of health-related activities on Second Life; to examine the design attributes of the most innovative and popular sites; and to assess the potential utility of Second Life for the dissemination of health information and for health behavior change. We used three separate search strategies to identify health-related sites on Second Life. The first used the application's search engine, entering both generic and select illness-specific keywords, to seek out sites. The second identified sites through a comprehensive review of print, blog, and media sources discussing health activities on Second Life. We then visited each site and used a snowball method to identify other health sites until we reached saturation (no new health sites were identified). The content, user experience, and chief purpose of each site were tabulated as well as basic site information, including user traffic data and site size. We found a wide range of health-related activities on Second Life, and a diverse group of users, including organizations, groups, and individuals. For many users, Second Life activities are a part of their Web 2.0 communication strategy. The most common type of health-related site in our sample (n = 68) were those whose principle aim was patient education or to increase awareness about health issues. The second most common type of site were support sites, followed by training sites, and marketing sites. Finally, a few sites were purpose-built to conduct research in SL or to recruit participants for real-life research. Studies show that behaviors from virtual worlds can translate to the real world. Our survey suggests that users are engaged in a range of health-related activities in Second Life which are potentially impacting real-life behaviors. Further research evaluating the impact of health-related activities on Second Life is warranted.

A Survey of Health-Related Activities on Second Life

PubMed Central

Beard, Leslie; Wilson, Kumanan; Morra, Dante

2009-01-01

Background Increasingly, governments, health care agencies, companies, and private groups have chosen Second Life as part of their Web 2.0 communication strategies. Second Life offers unique design features for disseminating health information, training health professionals, and enabling patient education for both academic and commercial health behavior research. Objectives This study aimed to survey and categorize the range of health-related activities on Second Life; to examine the design attributes of the most innovative and popular sites; and to assess the potential utility of Second Life for the dissemination of health information and for health behavior change. Methods We used three separate search strategies to identify health-related sites on Second Life. The first used the application’s search engine, entering both generic and select illness-specific keywords, to seek out sites. The second identified sites through a comprehensive review of print, blog, and media sources discussing health activities on Second Life. We then visited each site and used a snowball method to identify other health sites until we reached saturation (no new health sites were identified). The content, user experience, and chief purpose of each site were tabulated as well as basic site information, including user traffic data and site size. Results We found a wide range of health-related activities on Second Life, and a diverse group of users, including organizations, groups, and individuals. For many users, Second Life activities are a part of their Web 2.0 communication strategy. The most common type of health-related site in our sample (n = 68) were those whose principle aim was patient education or to increase awareness about health issues. The second most common type of site were support sites, followed by training sites, and marketing sites. Finally, a few sites were purpose-built to conduct research in SL or to recruit participants for real-life research. Conclusions Studies show that behaviors from virtual worlds can translate to the real world. Our survey suggests that users are engaged in a range of health-related activities in Second Life which are potentially impacting real-life behaviors. Further research evaluating the impact of health-related activities on Second Life is warranted. PMID:19632971

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases.

PubMed

Sugrue, Elena; Carr, Paul D; Scott, Colin; Jackson, Colin J

2016-11-15

The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK a of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

Crystal structure of a papain-fold protein without the catalytic residue: a novel member in the cysteine proteinase family.

PubMed

Zhang, Min; Wei, Zhiyi; Chang, Shaojie; Teng, Maikun; Gong, Weimin

2006-04-21

A 31kDa cysteine protease, SPE31, was isolated from the seeds of a legume plant, Pachyrizhus erosus. The protein was purified, crystallized and the 3D structure solved using molecular replacement. The cDNA was obtained by RT PCR followed by amplification using mRNA isolated from the seeds of the legume plant as a template. Analysis of the cDNA sequence and the 3D structure indicated the protein to belong to the papain family. Detailed analysis of the structure revealed an unusual replacement of the conserved catalytic Cys with Gly. Replacement of another conserved residue Ala/Gly by a Phe sterically blocks the access of the substrate to the active site. A polyethyleneglycol molecule and a natural peptide fragment were bound to the surface of the active site. Asn159 was found to be glycosylated. The SPE31 cDNA sequence shares several features with P34, a protein found in soybeans, that is implicated in plant defense mechanisms as an elicitor receptor binding to syringolide. P34 has also been shown to interact with vegetative storage proteins and NADH-dependent hydroxypyruvate reductase. These roles suggest that SPE31 and P34 form a unique subfamily within the papain family. The crystal structure of SPE31 complexed with a natural peptide ligand reveals a unique active site architecture. In addition, the clear evidence of glycosylated Asn159 provides useful information towards understanding the functional mechanism of SPE31/P34.

Biodiversity Sampling Using a Global Acoustic Approach: Contrasting Sites with Microendemics in New Caledonia

PubMed Central

Gasc, Amandine; Sueur, Jérôme; Pavoine, Sandrine; Pellens, Roseli; Grandcolas, Philippe

2013-01-01

New Caledonia is a Pacific island with a unique biodiversity showing an extreme microendemism. Many species distributions observed on this island are extremely restricted, localized to mountains or rivers making biodiversity evaluation and conservation a difficult task. A rapid biodiversity assessment method based on acoustics was recently proposed. This method could help to document the unique spatial structure observed in New Caledonia. Here, this method was applied in an attempt to reveal differences among three mountain sites (Mandjélia, Koghis and Aoupinié) with similar ecological features and species richness level, but with high beta diversity according to different microendemic assemblages. In each site, several local acoustic communities were sampled with audio recorders. An automatic acoustic sampling was run on these three sites for a period of 82 successive days. Acoustic properties of animal communities were analysed without any species identification. A frequency spectral complexity index (NP) was used as an estimate of the level of acoustic activity and a frequency spectral dissimilarity index (Df) assessed acoustic differences between pairs of recordings. As expected, the index NP did not reveal significant differences in the acoustic activity level between the three sites. However, the acoustic variability estimated by the index Df, could first be explained by changes in the acoustic communities along the 24-hour cycle and second by acoustic dissimilarities between the three sites. The results support the hypothesis that global acoustic analyses can detect acoustic differences between sites with similar species richness and similar ecological context, but with different species assemblages. This study also demonstrates that global acoustic methods applied at broad spatial and temporal scales could help to assess local biodiversity in the challenging context of microendemism. The method could be deployed over large areas, and could help to compare different sites and determine conservation priorities. PMID:23734245

Biodiversity sampling using a global acoustic approach: contrasting sites with microendemics in New Caledonia.

PubMed

Gasc, Amandine; Sueur, Jérôme; Pavoine, Sandrine; Pellens, Roseli; Grandcolas, Philippe

2013-01-01

New Caledonia is a Pacific island with a unique biodiversity showing an extreme microendemism. Many species distributions observed on this island are extremely restricted, localized to mountains or rivers making biodiversity evaluation and conservation a difficult task. A rapid biodiversity assessment method based on acoustics was recently proposed. This method could help to document the unique spatial structure observed in New Caledonia. Here, this method was applied in an attempt to reveal differences among three mountain sites (Mandjélia, Koghis and Aoupinié) with similar ecological features and species richness level, but with high beta diversity according to different microendemic assemblages. In each site, several local acoustic communities were sampled with audio recorders. An automatic acoustic sampling was run on these three sites for a period of 82 successive days. Acoustic properties of animal communities were analysed without any species identification. A frequency spectral complexity index (NP) was used as an estimate of the level of acoustic activity and a frequency spectral dissimilarity index (Df ) assessed acoustic differences between pairs of recordings. As expected, the index NP did not reveal significant differences in the acoustic activity level between the three sites. However, the acoustic variability estimated by the index Df , could first be explained by changes in the acoustic communities along the 24-hour cycle and second by acoustic dissimilarities between the three sites. The results support the hypothesis that global acoustic analyses can detect acoustic differences between sites with similar species richness and similar ecological context, but with different species assemblages. This study also demonstrates that global acoustic methods applied at broad spatial and temporal scales could help to assess local biodiversity in the challenging context of microendemism. The method could be deployed over large areas, and could help to compare different sites and determine conservation priorities.

Structure of granzyme C reveals an unusual mechanism of protease autoinhibition

PubMed Central

Kaiserman, Dion; Buckle, Ashley M.; Van Damme, Petra; Irving, James A.; Law, Ruby H. P.; Matthews, Antony Y.; Bashtannyk-Puhalovich, Tanya; Langendorf, Chris; Thompson, Philip; Vandekerckhove, Joël; Gevaert, Kris; Whisstock, James C.; Bird, Phillip I.

2009-01-01

Proteases act in important homeostatic pathways and are tightly regulated. Here, we report an unusual structural mechanism of regulation observed by the 2.5-Å X-ray crystal structure of the serine protease, granzyme C. Although the active-site triad residues adopt canonical conformations, the oxyanion hole is improperly formed, and access to the primary specificity (S1) pocket is blocked through a reversible rearrangement involving Phe-191. Specifically, a register shift in the 190-strand preceding the active-site serine leads to Phe-191 filling the S1 pocket. Mutation of a unique Glu–Glu motif at positions 192–193 unlocks the enzyme, which displays chymase activity, and proteomic analysis confirms that activity of the wild-type protease can be released through interactions with an appropriate substrate. The 2.5-Å structure of the unlocked enzyme reveals unprecedented flexibility in the 190-strand preceding the active-site serine that results in Phe-191 vacating the S1 pocket. Overall, these observations describe a broadly applicable mechanism of protease regulation that cannot be predicted by template-based modeling or bioinformatic approaches alone. PMID:19299505

Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa

PubMed Central

Morita, Hiroyuki; Wanibuchi, Kiyofumi; Nii, Hirohiko; Kato, Ryohei; Sugio, Shigetoshi; Abe, Ikuro

2010-01-01

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C6-C7-C6 diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C6-C3 coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H2O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C6-C7-C6 scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes. PMID:21041675

Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa.

PubMed

Morita, Hiroyuki; Wanibuchi, Kiyofumi; Nii, Hirohiko; Kato, Ryohei; Sugio, Shigetoshi; Abe, Ikuro

2010-11-16

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C(6)-C(7)-C(6) diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C(6)-C(3) coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H(2)O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C(6)-C(7)-C(6) scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes.

Revealing the Secrets of Stonehenge Through the Application of Laser Scanning, Photogrammetry and Visualisation Techniques

NASA Astrophysics Data System (ADS)

Bryan, P. G.; Abbott, M.; Dodson, A. J.

2013-07-01

Stonehenge is perhaps the most famous prehistoric monument in the world. Begun as a simple earthwork enclosure, it was built in several stages with the unique lintelled stone circle being erected in the Neolithic period in around 2,500 BC. Today Stonehenge, together with Avebury and other associated sites, form the heart of a World Heritage Site (WHS) with a unique and dense concentration of outstanding prehistoric monuments. In 2011 English Heritage (EH) embarked on a new survey of the monument. Undertaken by the Greenhatch Group, a commercial survey company based near Derby, a combination of laser scanning and photogrammetric approaches were used to generate the required scale and detailed level of output required by English Heritage. This paper will describe the background to this project and its context within previous survey activities at this World Heritage Site. It will explain the data acquisition technology and processes undertaken on site, the datasets derived from post-processing and their filtering and analysis within both subsequent research projects. Alongside a description of how the data is currently being exploited and proposed future applications within the conservation and management of the site, it will finish by considering the impact of developing geospatial imaging technologies.

Deconstructing thermodynamic parameters of a coupled system from site-specific observables.

PubMed

Chowdhury, Sandipan; Chanda, Baron

2010-11-02

Cooperative interactions mediate information transfer between structural domains of a protein molecule and are major determinants of protein function and modulation. The prevalent theories to understand the thermodynamic origins of cooperativity have been developed to reproduce the complex behavior of a global thermodynamic observable such as ligand binding or enzyme activity. However, in most cases the measurement of a single global observable cannot uniquely define all the terms that fully describe the energetics of the system. Here we establish a theoretical groundwork for analyzing protein thermodynamics using site-specific information. Our treatment involves extracting a site-specific parameter (defined as χ value) associated with a structural unit. We demonstrate that, under limiting conditions, the χ value is related to the direct interaction terms associated with the structural unit under observation and its intrinsic activation energy. We also introduce a site-specific interaction energy term (χ(diff)) that is a function of the direct interaction energy of that site with every other site in the system. When combined with site-directed mutagenesis and other molecular level perturbations, analyses of χ values of site-specific observables may provide valuable insights into protein thermodynamics and structure.

Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis

PubMed Central

Mancini, Marie; Machamer, Carolyn E.; Roy, Sophie; Nicholson, Donald W.; Thornberry, Nancy A.; Casciola-Rosen, Livia A.; Rosen, Antony

2000-01-01

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors. PMID:10791974

Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

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

Beres, Christopher M.; Fort, E. Joseph; Boyle, James D.

2013-07-01

The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an abovegroundmore » structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)« less

Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone.

PubMed

Rich, Joseph O; Budde, Cheryl L; McConeghey, Luke D; Cotterill, Ian C; Mozhaev, Vadim V; Singh, Sheo B; Goetz, Michael A; Zhao, Annie; Michels, Peter C; Khmelnitsky, Yuri L

2009-06-01

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.

Virus activated artificial ECM induces the osteoblastic differentiation of mesenchymal stem cells without osteogenic supplements

PubMed Central

Wang, Jianglin; Wang, Lin; Li, Xin; Mao, Chuanbin

2013-01-01

Biochemical and topographical features of an artificial extracellular matrix (aECM) can direct stem cell fate. However, it is difficult to vary only the biochemical cues without changing nanotopography to study their unique role. We took advantage of two unique features of M13 phage, a non-toxic nanofiber-like virus, to generate a virus-activated aECM with constant ordered ridge/groove nanotopography but displaying different fibronectin-derived peptides (RGD, its synergy site PHSRN, and a combination of RGD and PHSRN). One feature is the self-assembly of phage into a ridge/groove structure, another is the ease of genetically surface-displaying a peptide. We found that the unique ridge/groove nanotopography and the display of RGD and PHSRN could induce the osteoblastic differentiation of mesenchymal stem cells (MSCs) without any osteogenic supplements. The aECM formed through self-assembly and genetic engineering of phage can be used to understand the role of peptide cues in directing stem cell behavior while keeping nanotopography constant. PMID:23393624

Path to Collagenolysis

PubMed Central

Prior, Stephen H.; Byrne, Todd S.; Tokmina-Roszyk, Dorota; Fields, Gregg B.

2016-01-01

Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ∼1,080 Å2 of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile G∼VV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling. PMID:26887942

Cis-regulatory RNA elements that regulate specialized ribosome activity.

PubMed

Xue, Shifeng; Barna, Maria

2015-01-01

Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5'UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution.

The Root Transition Zone: A Hot Spot for Signal Crosstalk.

PubMed

Kong, Xiangpei; Liu, Guangchao; Liu, Jiajia; Ding, Zhaojun

2018-05-01

The root transition zone (TZ), located between the apical meristem and basal elongation region, has a unique role in root growth and development. The root TZ is not only the active site for hormone crosstalk, but also the perception site for various environmental cues, such as aluminum (Al) stress and low phosphate (Pi) stress. We propose that the root TZ is a hot spot for the integration of diverse inputs from endogenous (hormonal) and exogenous (sensorial) stimuli to control root growth. Copyright © 2018 Elsevier Ltd. All rights reserved.

Variations in protein/flavin hydrogen bonding in a LOV domain produce non-Arrhenius kinetics of adduct decay†

PubMed Central

Zoltowski, Brian D.; Nash, Abigail I.; Gardner, Kevin H.

2011-01-01

Light Oxygen Voltage (LOV) domains utilize a conserved blue light-dependent mechanism to control a diverse array of effector domains in biological and engineered proteins. Variations in the kinetics and efficiency of LOV photochemistry fine tune various aspects of the photic response. Characterization of the kinetics of a key aspect of this photochemical mechanism in EL222, a blue-light responsive DNA binding protein from Erythrobacter litoralis HTCC2594, reveals unique non-Arrhenius behavior in the rate of dark state cleavage of the photochemically-generated adduct. Sequence analysis and mutagenesis studies establish that this effect stems from a Gln to Ala mutation unique to EL222 and homologous proteins from marine bacteria. Kinetic and spectroscopic analyses reveal that hydrogen bonding interactions between the FMN N1, O2 and ribityl hydroxyls with the surrounding protein regulate photocycle kinetics and stabilize the LOV active site from temperature-induced alteration in local structure. Substitution of residues interacting with the N1-O2 locus modulates adduct stability, structural flexibility and sequestration of the active site from bulk solvent without perturbation of light-activated DNA binding. Together, these variants link non-Arrhenius behavior to specific alteration of an H-bonding network, while affording tunability of photocycle kinetics. PMID:21923139

Variations in protein-flavin hydrogen bonding in a light, oxygen, voltage domain produce non-Arrhenius kinetics of adduct decay.

PubMed

Zoltowski, Brian D; Nash, Abigail I; Gardner, Kevin H

2011-10-18

Light, oxygen, voltage (LOV) domains utilize a conserved blue light-dependent mechanism to control a diverse array of effector domains in biological and engineered proteins. Variations in the kinetics and efficiency of LOV photochemistry fine-tune various aspects of the photic response. Characterization of the kinetics of a key aspect of this photochemical mechanism in EL222, a blue light responsive DNA binding protein from Erythrobacter litoralis HTCC2594, reveals unique non-Arrhenius behavior in the rate of dark-state cleavage of the photochemically generated adduct. Sequence analysis and mutagenesis studies establish that this effect stems from a Gln to Ala mutation unique to EL222 and homologous proteins from marine bacteria. Kinetic and spectroscopic analyses reveal that hydrogen bonding interactions between the FMN N1, O2, and ribityl hydroxyls and the surrounding protein regulate photocycle kinetics and stabilize the LOV active site from temperature-induced alteration in local structure. Substitution of residues interacting with the N1-O2 locus modulates adduct stability, structural flexibility, and sequestration of the active site from bulk solvent without perturbation of light-activated DNA binding. Together, these variants link non-Arrhenius behavior to specific alteration of an H-bonding network, while affording tunability of photocycle kinetics. © 2011 American Chemical Society

Automethylation of Protein Arginine Methyltransferase 8 (PRMT8) Regulates Activity by Impeding S-Adenosylmethionine Sensitivity*

PubMed Central

Dillon, Myles B. C.; Rust, Heather L.; Thompson, Paul R.; Mowen, Kerri A.

2013-01-01

Protein arginine methyltransferase (PRMT) 8 is unique among the PRMTs, as it has a highly restricted tissue expression pattern and an N terminus that contains two automethylation sites and a myristoylation site. PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to a peptidylarginine on a protein substrate. Currently, the physiological roles, regulation, and cellular substrates of PRMT8 are poorly understood. However, a thorough understanding of PRMT8 kinetics should provide insights into each of these areas, thereby enhancing our understanding of this unique enzyme. In this study, we determined how automethylation regulates the enzymatic activity of PRMT8. We found that preventing automethylation with lysine mutations (preserving the positive charge of the residue) increased the turnover rate and decreased the Km of AdoMet but did not affect the Km of the protein substrate. In contrast, mimicking automethylation with phenylalanine (i.e. mimicking the increased hydrophobicity) decreased the turnover rate. The inhibitory effect of the PRMT8 N terminus could be transferred to PRMT1 by creating a chimeric protein containing the N terminus of PRMT8 fused to PRMT1. Thus, automethylation of the N terminus likely regulates PRMT8 activity by decreasing the affinity of the enzyme for AdoMet. PMID:23946480

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

DOE PAGES

Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; ...

2015-12-17

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternarymore » structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. As a result, the active site architecture shows clear evidence of having arisen by convergent evolution.« less

Design of potent and selective human cathepsin K inhibitors that span the active site

PubMed Central

Thompson, Scott K.; Halbert, Stacie M.; Bossard, Mary J.; Tomaszek, Thaddeus A.; Levy, Mark A.; Zhao, Baoguang; Smith, Ward W.; Abdel-Meguid, Sherin S.; Janson, Cheryl A.; D’Alessio, Karla J.; McQueney, Michael S.; Amegadzie, Bernard Y.; Hanning, Charles R.; DesJarlais, Renee L.; Briand, Jacques; Sarkar, Susanta K.; Huddleston, Michael J.; Ijames, Carl F.; Carr, Steven A.; Garnes, Keith T.; Shu, Art; Heys, J. Richard; Bradbeer, Jeremy; Zembryki, Denise; Lee-Rykaczewski, Liz; James, Ian E.; Lark, Michael W.; Drake, Fred H.; Gowen, Maxine; Gleason, John G.; Veber, Daniel F.

1997-01-01

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention. PMID:9405598

Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

PubMed

Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

2016-03-24

Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the -2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the -1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes.

HHM motif at the CuH-site of peptidylglycine monooxygenase is a pH-dependent conformational switch.

PubMed

Kline, Chelsey D; Mayfield, Mary; Blackburn, Ninian J

2013-04-16

Peptidylglycine monooxygenase is a copper-containing enzyme that catalyzes the amidation of neuropeptides hormones, the first step of which is the conversion of a glycine-extended pro-peptide to its α-hydroxyglcine intermediate. The enzyme contains two mononuclear Cu centers termed CuM (ligated to imidazole nitrogens of H242, H244 and the thioether S of M314) and CuH (ligated to imidazole nitrogens of H107, H108, and H172) with a Cu-Cu separation of 11 Å. During catalysis, the M site binds oxygen and substrate, and the H site donates the second electron required for hydroxylation. The WT enzyme shows maximum catalytic activity at pH 5.8 and undergoes loss of activity at lower pHs due to a protonation event with a pKA of 4.6. Low pH also causes a unique structural transition in which a new S ligand coordinates to copper with an identical pKA, manifest by a large increase in Cu-S intensity in the X- ray absorption spectroscopy. In previous work (Bauman, A. T., Broers, B. A., Kline, C. D., and Blackburn, N. J. (2011) Biochemistry 50, 10819-10828), we tentatively assigned the new Cu-S interaction to binding of M109 to the H-site (part of an HHM conserved motif common to all but one member of the family). Here we follow up on these findings via studies on the catalytic activity, pH-activity profiles, and spectroscopic (electron paramagnetic resonance, XAS, and Fourier transform infrared) properties of a number of H-site variants, including H107A, H108A, H172A, and M109I. Our results establish that M109 is indeed the coordinating ligand and confirm the prediction that the low pH structural transition with associated loss of activity is abrogated when the M109 thioether is absent. The histidine mutants show more complex behavior, but the almost complete lack of activity in all three variants coupled with only minor differences in their spectroscopic properties suggests that unique structural elements at H are critical for functionality. The data suggest a more general utility for the HHM motif as a copper- and pH-dependent conformational switch.

Influence of iron doping on tetravalent nickel content in catalytic oxygen evolving films

PubMed Central

Li, Nancy; Bediako, D. Kwabena; Hadt, Ryan G.; Hayes, Dugan; Kempa, Thomas J.; von Cube, Felix; Bell, David C.; Chen, Lin X.; Nocera, Daniel G.

2017-01-01

Iron doping of nickel oxide films results in enhanced activity for promoting the oxygen evolution reaction (OER). Whereas this enhanced activity has been ascribed to a unique iron site within the nickel oxide matrix, we show here that Fe doping influences the Ni valency. The percent of Fe3+ doping promotes the formation of formal Ni4+, which in turn directly correlates with an enhanced activity of the catalyst in promoting OER. The role of Fe3+ is consistent with its behavior as a superior Lewis acid. PMID:28137835

Influence of iron doping on tetravalent nickel content in catalytic oxygen evolving films

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

Li, Nancy; Bediako, D. Kwabena; Hadt, Ryan G.

2017-01-30

Iron doping of nickel oxide films results in enhanced activity for promoting the oxygen evolution reaction (OER). Whereas this enhanced activity has been ascribed to a unique iron site within the nickel oxide matrix, we show here that Fe doping influences the Ni valency. The percent of Fe3+ doping promotes the formation of formal Ni4+, which in turn directly correlates with an enhanced activity of the catalyst in promoting OER. The role of Fe3+ is consistent with its behavior as a superior Lewis acid.

Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.

PubMed

van Kasteren, Puck B; Bailey-Elkin, Ben A; James, Terrence W; Ninaber, Dennis K; Beugeling, Corrine; Khajehpour, Mazdak; Snijder, Eric J; Mark, Brian L; Kikkert, Marjolein

2013-02-26

Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc-dependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-β mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual-specificity proteases.

Tuning a Protein-Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation.

PubMed

Pham, Grace H; Ou, Weijia; Bursulaya, Badry; DiDonato, Michael; Herath, Ananda; Jin, Yunho; Hao, Xueshi; Loren, Jon; Spraggon, Glen; Brock, Ansgar; Uno, Tetsuo; Geierstanger, Bernhard H; Cellitti, Susan E

2018-04-16

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Drosophila serpin 4 functions as a neuroserpin-like inhibitor of subtilisin-like proprotein convertases.

PubMed

Osterwalder, Thomas; Kuhnen, Angela; Leiserson, William M; Kim, You-Seung; Keshishian, Haig

2004-06-16

The proteolytic processing of neuropeptide precursors is believed to be regulated by serine proteinase inhibitors, or serpins. Here we describe the molecular cloning and functional expression of a novel member of the serpin family, Serine protease inhibitor 4 (Spn4), that we propose is involved in the regulation of peptide maturation in Drosophila. The Spn4 gene encodes at least two different serpin proteins, generated by alternate splicing of the last coding exon. The closest vertebrate homolog to Spn4 is neuroserpin. Like neuroserpin, one of the Spn4 proteins (Spn4.1) features a unique C-terminal extension, reminiscent of an endoplasmic reticulum (ER) retention signal; however, Spn4.1 and neuroserpin have divergent reactive site loops, with Spn4.1 showing a generic recognition site for furin/SPC1, the founding member of the intracellularly active family of subtilisin-like proprotein convertases (SPCs). In vitro, Spn4.1 forms SDS-stable complexes with the SPC furin and directly inhibits it. When Spn4.1 is overexpressed in specific peptidergic cells of Drosophila larvae, the animals exhibit a phenotype consistent with disrupted neuropeptide processing. This observation, together with the unique combination of an ER-retention signal, a target sequence for SPCs in the reactive site loop, and the in vitro inhibitory activity against furin, strongly suggests that Spn4.1 is an intracellular regulator of SPCs.

Nurses Improving the Care of Healthsystem Elders: creating a sustainable business model to improve care of hospitalized older adults.

PubMed

Capezuti, Elizabeth A; Bricoli, Barbara; Briccoli, Barbara; Boltz, Marie P

2013-08-01

The Nurses Improving the Care of Healthsystem Elders (NICHE) program helps its more than 450 member sites to build the leadership capabilities to enact system-level change that targets the unique needs of older adults and embeds evidence-based geriatrics knowledge into practice. NICHE received expansion funding to establish a sustainable business model for operations while positioning the program to continue as a leader in innovative senior care programs. The expansion program focused on developing an internal business infrastructure, expanding NICHE-specific resources, creating a Web platform, increasing the number of participating NICHE hospitals, enhancing and expanding the NICHE benchmarking service, supporting research that generates evidence-based practices, fostering interorganizational collaboration, developing sufficient diversified revenue sources, and increasing the penetration and level of activity of current NICHE sites. These activities (improved services, Web-based tools, better benchmarking) added value and made it feasible to charge hospitals an annual fee for access and participation. NICHE does not stipulate how institutions should modify geriatric care; rather, NICHE principles and tools are meant to be adapted to each site's unique institutional culture. This article describes the historical context, the rationale, and the business plan that has resulted in successful organizational outcomes, including financial sustainability of the business operations of NICHE. © 2013, Copyright the Authors Journal compilation © 2013, The American Geriatrics Society.

Discussion of impact of relics activation on protection and utilization approaches-take the old summer palace as an example

NASA Astrophysics Data System (ADS)

Xiaoqi, J.

2015-08-01

As the popularization of cultural relics and the rapid development of cultural tourism industry, a large number of cultural relic tourism resources goes into public eyes. Activation of relics has became an important way for tourist to contact and understand culture relics. The way of how to properly interpret the historical sense and cultural uniqueness to the masses of tourists in order to achieve social service functions of relic resources has always been research focal point of site protection and utilization, so nowadays it has important significance to protection and utilization of heritage resources in our country. From the point of activation of relics and based on the analysis of resource characteristic, the paper in depth discuss ways of activation of relics of the Old Summer Palace, in order to provide reference for sustainable development of sites tourism in China.

[NiFe] hydrogenases: a common active site for hydrogen metabolism under diverse conditions.

PubMed

Shafaat, Hannah S; Rüdiger, Olaf; Ogata, Hideaki; Lubitz, Wolfgang

2013-01-01

Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. The most abundant hydrogenases contain a [NiFe] active site; these proteins are generally biased towards hydrogen oxidation activity and are reversibly inhibited by oxygen. However, there are [NiFe] hydrogenase that exhibit unique properties, including aerobic hydrogen oxidation and preferential hydrogen production activity; these proteins are highly relevant in the context of biotechnological devices. This review describes four classes of these "nonstandard" [NiFe] hydrogenases and discusses the electrochemical, spectroscopic, and structural studies that have been used to understand the mechanisms behind this exceptional behavior. A revised classification protocol is suggested in the conclusions, particularly with respect to the term "oxygen-tolerance". This article is part of a special issue entitled: metals in bioenergetics and biomimetics systems. Copyright © 2013 Elsevier B.V. All rights reserved.

A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.

PubMed

Kudalkar, Shalley N; Njuma, Olive J; Li, Yongjiang; Muldowney, Michelle; Fuanta, N Rene; Goodwin, Douglas C

2015-03-03

Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris.

PubMed

Lei, Da; Xu, Yang; He, Qinghua; Pang, Yifeng; Chen, Bo; Xiong, Liang; Li, Yanping

2013-12-01

Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff's base staining and Endo H digestion. Moreover, the deglycosylated protein's molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI-TOF-MS, and the hyperglycosylation extent was 21 %. The N-glycosylation site of rNPI was analyzed by nano LC-MS/MS after digesting by trypsin and Glu-C, and the unique potential site Asn41 of mature peptide was found to be glycosylated. Homology modeling of the 3D structure of rNPI indicated that the attached N-glycans hardly affected neutral protease's activity due to the great distance away from the active site of the enzyme.

Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide

PubMed Central

Thomas, Elizabeth A.; Carson, Monica J.; Neal, Michael J.; Sutcliffe, J. Gregor

1997-01-01

The effects of oleamide, an amidated lipid isolated from the cerebrospinal fluid of sleep-deprived cats, on serotonin receptor-mediated responses were investigated in cultured mammalian cells. In rat P11 cells, which endogenously express the 5-hydroxytryptamine2A (5HT2A) receptor, oleamide significantly potentiated 5HT-induced phosphoinositide hydrolysis. In HeLa cells expressing the 5HT7 receptor subtype, oleamide caused a concentration-dependent increase in cAMP accumulation but with lower efficacy than that observed by 5HT. This effect was not observed in untransfected HeLa cells. Clozapine did not prevent the increase in cAMP elicited by oleamide, and ketanserin caused an ≈65% decrease. In the presence of 5HT, oleamide had the opposite effect on cAMP, causing insurmountable antagonism of the concentration-effect curve to 5HT, but had no effect on cAMP levels elicited by isoproterenol or forskolin. These results indicate that oleamide can modulate 5HT-mediated signal transduction at different subtypes of mammalian 5HT receptors. Additionally, our data indicate that oleamide acts at an apparent allosteric site on the 5HT7 receptor and elicits functional responses via activation of this site. This represents a unique mechanism of activation for 5HT G protein-coupled receptors and suggests that G protein-coupled neurotransmitter receptors may act like their iontropic counterparts (i.e., γ-aminobutyric acid type A receptors) in that there may be several binding sites on the receptor that regulate functional activity with varying efficacies. PMID:9391162

AccrualNet: Addressing Low Accrual Via a Knowledge-Based, Community of Practice Platform

PubMed Central

Massett, Holly A.; Parreco, Linda K.; Padberg, Rose Mary; Richmond, Ellen S.; Rienzo, Marie E.; Leonard, Colleen E. Ryan; Quesenbery, Whitney; Killiam, H. William; Johnson, Lenora E.; Dilts, David M.

2011-01-01

Purpose: Present the design and initial evaluation of a unique, Web-enabled platform for the development of a community of practice around issues of oncology clinical trial accrual. Methods: The National Cancer Institute (NCI) conducted research with oncology professionals to identify unmet clinical trial accrual needs in the field. In response, a comprehensive platform for accrual resources, AccrualNet, was created by using an agile development process, storyboarding, and user testing. Literature and resource searches identified relevant content to populate the site. Descriptive statistics were tracked for resource and site usage. Use cases were defined to support implementation. Results: AccrualNet has five levels: (1) clinical trial macrostages (prestudy, active study, and poststudy); (2) substages (developing a protocol, selecting a trial, preparing to open, enrolling patients, managing the trial, retaining participants, and lessons learned); (3) strategies for each substage; (4) multiple activities for each strategy; and (5) multiple resources for each activity. Since its launch, AccrualNet has had more than 45,000 page views, with the Tools & Resources, Conversations, and Training sections being the most viewed. Total resources have increased 69%, to 496 items. Analysis of articles in the site reveals that 22% are from two journals and 46% of the journals supplied a single article. To date, there are 29 conversations with 43 posts. Four use cases are discussed. Conclusion: AccrualNet represents a unique, centralized comprehensive-solution platform to systematically capture accrual knowledge for all stages of a clinical trial. It is designed to foster a community of practice by encouraging users to share additional strategies, resources, and ideas. PMID:22379429

AccrualNet: Addressing Low Accrual Via a Knowledge-Based, Community of Practice Platform.

PubMed

Massett, Holly A; Parreco, Linda K; Padberg, Rose Mary; Richmond, Ellen S; Rienzo, Marie E; Leonard, Colleen E Ryan; Quesenbery, Whitney; Killiam, H William; Johnson, Lenora E; Dilts, David M

2011-11-01

Present the design and initial evaluation of a unique, Web-enabled platform for the development of a community of practice around issues of oncology clinical trial accrual. The National Cancer Institute (NCI) conducted research with oncology professionals to identify unmet clinical trial accrual needs in the field. In response, a comprehensive platform for accrual resources, AccrualNet, was created by using an agile development process, storyboarding, and user testing. Literature and resource searches identified relevant content to populate the site. Descriptive statistics were tracked for resource and site usage. Use cases were defined to support implementation. ACCRUALNET HAS FIVE LEVELS: (1) clinical trial macrostages (prestudy, active study, and poststudy); (2) substages (developing a protocol, selecting a trial, preparing to open, enrolling patients, managing the trial, retaining participants, and lessons learned); (3) strategies for each substage; (4) multiple activities for each strategy; and (5) multiple resources for each activity. Since its launch, AccrualNet has had more than 45,000 page views, with the Tools & Resources, Conversations, and Training sections being the most viewed. Total resources have increased 69%, to 496 items. Analysis of articles in the site reveals that 22% are from two journals and 46% of the journals supplied a single article. To date, there are 29 conversations with 43 posts. Four use cases are discussed. AccrualNet represents a unique, centralized comprehensive-solution platform to systematically capture accrual knowledge for all stages of a clinical trial. It is designed to foster a community of practice by encouraging users to share additional strategies, resources, and ideas.

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

Taneja, Bhupesh; Patel, Asmita; Slesarev, Alexei

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting nomore » conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.« less

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration*

PubMed Central

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W.

2014-01-01

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions. PMID:24532791

Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

PubMed

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W

2014-03-28

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

Sulfur reduction in sediments of marine and evaporite environments

NASA Technical Reports Server (NTRS)

Klug, M. J.; Boston, P.; Francois, R.; Gyure, R. A.; Javor, B.; Tribble, G.; Vairavamurthy, A.

1985-01-01

Transformations of sulfur in sediments of ponds ranging in salinities from that of normal seawater to those of brines saturated with sodium chloride were examined. The chemistry of the sediment and pore waters were focused on with emphasis on the fate of sulfate reduction. The effects of increasing salinity on both forms of sulfur and microbial activity were determined. A unique set of chemical profiles and sulfate-reducing activity was found for the sediments of each of the sites examined. The quantity of organic matter in the salt pond sediments was significantly greater than that occurring in the adjacent intertidal site. The total quantitative and qualitative distribution of volatile fatty acids was also greater in the salt ponds. Volatile fatty acids increased with salinity.

Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies

PubMed Central

Rajan, Rakhi; Prasad, Rajendra; Taneja, Bhupesh; Wilson, Samuel H.; Mondragón, Alfonso

2013-01-01

Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo. PMID:23125368

EPA Region 2 SEMS_CERCLIS Sites All [R2] and SEMS_CERCLIS Sites NPL [R2] GIS Layers

EPA Pesticide Factsheets

The Region 2 SEMS_CERCLIS Sites All [R2] GIS layer contains unique Superfund Enterprise Management System (SEMS) site records. These records have the following NPL_STATUS designations: CURRENTLY ON FINAL NPL, DELETED FROM FINAL NPL, NOT ON NPL, PROPOSED FOR NPL, REMOVED FROM PROPOSED NPL, and SITE IS PART OF NPL SITE. The Region 2 SEMS_CERCLIS NPL Sites [R2] GIS layer only has SEMS records with the following NPL_STATUS designations: 'CURRENTLY ON FINAL NPL', 'DELETED FROM FINAL NPL', 'PROPOSED FOR NPL'.The Superfund Enterprise Management System (SEMS) is EPA's official record for tracking hazardous waste sites, potentially hazardous waste sites, and remedial activities performed in support of the Superfund Program across the nation. This includes sites that are on the National Priorities List (NPL) or are being considered for the NPL. SEMS represents a joint development and ongoing collaboration between Superfund's Remedial, Removal, Federal Facilities, Enforcement, and Emergency Response programs. It provides its wide audience base with a means of ongoing analysis of Superfund Program activities and informational needs at the site, regional management, and national management levels. The customers of SEMS or SEMS data are five EPA Headquarters offices and regional staff, citizens, the regulated community, other Federal agencies, States, Tribes, local agencies, and industry. SEMS stakeholders are States, Congress, other Federal agencies, industry groups, and cit

Online social networks that connect users to physical activity partners: a review and descriptive analysis.

PubMed

Nakhasi, Atul; Shen, Album Xiaotian; Passarella, Ralph Joseph; Appel, Lawrence J; Anderson, Cheryl Am

2014-06-16

The US Centers for Disease Control and Prevention have identified a lack of encouragement, support, or companionship from family and friends as a major barrier to physical activity. To overcome this barrier, online social networks are now actively leveraging principles of companion social support in novel ways. The aim was to evaluate the functionality, features, and usability of existing online social networks which seek to increase physical activity and fitness among users by connecting them to physical activity partners, not just online, but also face-to-face. In September 2012, we used 3 major databases to identify the website addresses for relevant online social networks. We conducted a Google search using 8 unique keyword combinations: the common keyword "find" coupled with 1 of 4 prefix terms "health," "fitness," "workout," or "physical" coupled with 1 of 2 stem terms "activity partners" or "activity buddies." We also searched 2 prominent technology start-up news sites, TechCrunch and Y Combinator, using 2 unique keyword combinations: the common keyword "find" coupled with 1 of 2 stem terms "activity partners" and "activity buddies." Sites were defined as online social health activity networks if they had the ability to (1) actively find physical activity partners or activities for the user, (2) offer dynamic, real-time tracking or sharing of social activities, and (3) provide virtual profiles to users. We excluded from our analysis sites that were not Web-based, publicly available, in English, or free. Of the 360 initial search results, we identified 13 websites that met our complete criteria of an online social health activity network. Features such as physical activity creation (13/13, 100%) and private messaging (12/13, 92%) appeared almost universally among these websites. However, integration with Web 2.0 technologies such as Facebook and Twitter (9/13, 69%) and the option of direct event joining (8/13, 62%) were not as universally present. Largely absent were more sophisticated features that would enable greater usability, such as interactive engagement prompts (3/13, 23%) and system-created best fit activities (3/13, 23%). Several major online social networks that connect users to physical activity partners currently exist and use standardized features to achieve their goals. Future research is needed to better understand how users utilize these features and how helpful they truly are.

Factors that attract and repel visitation to urban recreation sites: a framework for research

Treesearch

David Klenosky; Cherie LeBlanc; Christine Vogt; Herbert Schroeder

2008-01-01

The mix of natural features and manmade elements in urban and metropolitan areas presents unique challenges for resource managers and planners. While some elements of the urban landscape (e.g., forested areas, parks, water features, and museums) may attract or encourage visitation, others (e.g., industrial and commercial activity, odors, noises, crime, litter, and...

Lessons for Higher Education: The University as a Site of Activism

ERIC Educational Resources Information Center

Lynch, Kathleen

2010-01-01

Len Barton is acutely aware of the power of the academy to either enhance critical thinking or to depress it. He is a true academic, never accepting the received wisdom or perspective of any given sociological standpoint, no matter how powerful or fashionable it was at the time. He has encouraged and promoted a unique blend of professional and…

Relationship between Use of Online Support Materials and Student Performance in an Introductory Finance Course

ERIC Educational Resources Information Center

Biktimirov, Ernest N.; Klassen, Kenneth J.

2008-01-01

The authors examined the relationship between student online activity, including access to specific course materials, and performance in a traditional face-to-face introductory finance course that a class Web site supported. The authors used 6 measures: (a) total hits, (b) hit consistency, (c) number of unique files that the students accessed, (d)…

Brain Region-Specific Activity Patterns after Recent or Remote Memory Retrieval of Auditory Conditioned Fear

ERIC Educational Resources Information Center

Kwon, Jeong-Tae; Jhang, Jinho; Kim, Hyung-Su; Lee, Sujin; Han, Jin-Hee

2012-01-01

Memory is thought to be sparsely encoded throughout multiple brain regions forming unique memory trace. Although evidence has established that the amygdala is a key brain site for memory storage and retrieval of auditory conditioned fear memory, it remains elusive whether the auditory brain regions may be involved in fear memory storage or…

Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives.

PubMed

Oyama, Takahiro; Yoshimori, Atsushi; Takahashi, Satoshi; Yamamoto, Tetsuya; Sato, Akira; Kamiya, Takanori; Abe, Hideaki; Abe, Takehiko; Tanuma, Sei-Ichi

2017-07-01

So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4'-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4'-hydroxylation and further decreased by 4'-methoxylation against mushroom tyrosinase. Surprisingly, 4'-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4'-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4'-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Social Inquiry and Social Action: Priorities for Preparing School Leaders

ERIC Educational Resources Information Center

Gale, Trevor

2010-01-01

Schools are uniquely placed in democracies. Among other things, they are sites of learning about things democratic, including learning "through" as one (important) way of learning "about." There are other sites in which to learn about and through democracies but schooling's uniqueness is that it is a site through which all must pass. So…

Quantification of pancreatic cancer proteome and phosphorylome: indicates molecular events likely contributing to cancer and activity of drug targets.

PubMed

Britton, David; Zen, Yoh; Quaglia, Alberto; Selzer, Stefan; Mitra, Vikram; Löβner, Christopher; Jung, Stephan; Böhm, Gitte; Schmid, Peter; Prefot, Petra; Hoehle, Claudia; Koncarevic, Sasa; Gee, Julia; Nicholson, Robert; Ward, Malcolm; Castellano, Leandro; Stebbing, Justin; Zucht, Hans Dieter; Sarker, Debashis; Heaton, Nigel; Pike, Ian

2014-01-01

LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application. Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset. Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power. Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.

A Rigidifying Salt-Bridge Favors the Activity of Thermophilic Enzyme at High Temperatures at the Expense of Low-Temperature Activity

PubMed Central

Lam, Sonia Y.; Yeung, Rachel C. Y.; Yu, Tsz-Ha; Sze, Kong-Hung; Wong, Kam-Bo

2011-01-01

Background Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity. Methods and Findings Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy. Conclusions Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures. PMID:21423654

A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.

PubMed

Lam, Sonia Y; Yeung, Rachel C Y; Yu, Tsz-Ha; Sze, Kong-Hung; Wong, Kam-Bo

2011-03-01

Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity. Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy. Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

Site Formation Processes and Hunter-Gatherers Use of Space in a Tropical Environment: A Geo-Ethnoarchaeological Approach from South India

PubMed Central

Friesem, David E.; Lavi, Noa; Madella, Marco; Ajithprasad, P.; French, Charles

2016-01-01

Hunter-gatherer societies have distinct social perceptions and practices which are expressed in unique use of space and material deposition patterns. However, the identification of archaeological evidence associated with hunter-gatherer activity is often challenging, especially in tropical environments such as rainforests. We present an integrated study combining ethnoarchaeology and geoarchaeology in order to study archaeological site formation processes related to hunter-gatherers’ ways of living in tropical forests. Ethnographic data was collected from an habitation site of contemporary hunter-gatherers in the forests of South India, aimed at studying how everyday activities and way of living dictate patterns of material deposition. Ethnoarchaeological excavations of abandoned open-air sites and a rock-shelter of the same group located deep in the forests, involved field observations and sampling of sediments from the abandoned sites and the contemporary site. Laboratory analyses included geochemical analysis (i.e., FTIR, ICP-AES), phytolith concentration analysis and soil micromorphology. The results present a dynamic spatial deposition pattern of macroscopic, microscopic and chemical materials, which stem from the distinctive ways of living and use of space by hunter-gatherers. This study shows that post-depositional processes in tropical forests result in poor preservation of archaeological materials due to acidic conditions and intensive biological activity within the sediments. Yet, the multiple laboratory-based analyses were able to trace evidence for activity surfaces and their maintenance practices as well as localized concentrations of activity remains such as the use of plants, metals, hearths and construction materials. PMID:27783683

Binding site in eag voltage sensor accommodates a variety of ions and is accessible in closed channel.

PubMed

Silverman, William R; Bannister, John P A; Papazian, Diane M

2004-11-01

In ether-a-go-go K+ channels, voltage-dependent activation is modulated by ion binding to a site located in an extracellular-facing crevice between transmembrane segments S2 and S3 in the voltage sensor. We find that acidic residues D278 in S2 and D327 in S3 are able to coordinate a variety of divalent cations, including Mg2+, Mn2+, and Ni2+, which have qualitatively similar functional effects, but different half-maximal effective concentrations. Our data indicate that ions binding to individual voltage sensors in the tetrameric channel act without cooperativity to modulate activation gating. We have taken advantage of the unique phenotype of Ni2+ in the D274A channel, which contains a mutation of a nonbinding site residue, to demonstrate that ions can access the binding site from the extracellular solution when the voltage sensor is in the resting conformation. Our results are difficult to reconcile with the x-ray structure of the KvAP K+ channel, in which the binding site residues are widely separated, and with the hydrophobic paddle model for voltage-dependent activation, in which the voltage sensor domain, including the S3-S4 loop, is near the cytoplasmic side of the membrane in the closed channel.

Spontaneous and evoked release are independently regulated at individual active zones.

PubMed

Melom, Jan E; Akbergenova, Yulia; Gavornik, Jeffrey P; Littleton, J Troy

2013-10-30

Neurotransmitter release from synaptic vesicle fusion is the fundamental mechanism for neuronal communication at synapses. Evoked release following an action potential has been well characterized for its function in activating the postsynaptic cell, but the significance of spontaneous release is less clear. Using transgenic tools to image single synaptic vesicle fusion events at individual release sites (active zones) in Drosophila, we characterized the spatial and temporal dynamics of exocytotic events that occur spontaneously or in response to an action potential. We also analyzed the relationship between these two modes of fusion at single release sites. A majority of active zones participate in both modes of fusion, although release probability is not correlated between the two modes of release and is highly variable across the population. A subset of active zones is specifically dedicated to spontaneous release, indicating a population of postsynaptic receptors is uniquely activated by this mode of vesicle fusion. Imaging synaptic transmission at individual release sites also revealed general rules for spontaneous and evoked release, and indicate that active zones with similar release probability can cluster spatially within individual synaptic boutons. These findings suggest neuronal connections contain two information channels that can be spatially segregated and independently regulated to transmit evoked or spontaneous fusion signals.

Microtubule-stabilizing properties of the avocado-derived toxins (+)-(R)-persin and (+)-(R)-tetrahydropersin in cancer cells and activity of related synthetic analogs.

PubMed

Field, Jessica J; Kanakkanthara, Arun; Brooke, Darby G; Sinha, Saptarshi; Pillai, Sushila D; Denny, William A; Butt, Alison J; Miller, John H

2016-06-01

The avocado toxin (+)-R-persin (persin) is active at low micromolar concentrations against breast cancer cells and synergizes with the estrogen receptor modulator 4-hydroxytamoxifen. Previous studies in the estrogen receptor-positive breast cancer cell line MCF-7 indicate that persin acts as a microtubule-stabilizing agent. In the present study, we further characterize the properties of persin and several new synthetic analogues in human ovarian cancer cells. Persin and tetrahydropersin cause G2M cell cycle arrest and increase intracellular microtubule polymerization. One analog (4-nitrophenyl)-deshydroxypersin prevents cell proliferation and blocks cells in G1 of the cell cycle rather than G2M, suggesting an additional mode of action of these compounds independent of microtubules. Persin can synergize with other microtubule-stabilizing agents, and is active against cancer cells that overexpress the P-glycoprotein drug efflux pump. Evidence from Flutax-1 competition experiments suggests that while the persin binding site on β-tubulin overlaps the classical taxoid site where paclitaxel and epothilone bind, persin retains activity in cell lines with single amino acid mutations that affect these other taxoid site ligands. This implies the existence of a unique binding location for persin at the taxoid site.

Viral proteases: an emerging therapeutic target.

PubMed

Korant, B D

1988-01-01

Only a few viral diseases are presently treatable because of our limited knowledge of specific viral target molecules. An attractive class of viral molecules toward which chemotherapeutic agents could be aimed are proteases coded by some virus groups such as retro- or picornaviruses (poliomyelitis, common cold virus). The picornavirus enzymes were discovered first, and they have now been characterized by a combination of molecular-genetic and biochemical approaches. Several laboratories have expressed the picornaviral enzymes in heterologous systems and have reported proteolytic activity, as well as the high cleavage fidelity diagnostic of the viral proteases. After dealing with several technical difficulties often encountered in standard genetic engineering approaches, one viral protease is now available to us in quantity and is amendable to mutagenic procedures. The initial outcome of the mutagenesis studies has been the confirmation of our earlier work with inhibitors, which suggested a cysteine active-site class. There is a clustering of active-site residues which may be unique to these viruses. The requirement for an active-site cysteine-histidine pair in combination with detailed information on the viral cleavage sites has permitted design of selective inhibitors with attractive antiviral properties. Future goals include investigation of the structural basis for selective processing and application of the cleavage specificity to general problems in genetic engineering.

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem.

PubMed Central

Ananvoranich, S; Lafontaine, D A; Perreault, J P

1999-01-01

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway. PMID:10037808

KatB, a cyanobacterial Mn-catalase with unique active site configuration: Implications for enzyme function.

PubMed

Bihani, Subhash C; Chakravarty, Dhiman; Ballal, Anand

2016-04-01

Manganese catalases (Mn-catalases), a class of H2O2 detoxifying proteins, are structurally and mechanistically distinct from the commonly occurring catalases, which contain heme. Active site of Mn-catalases can serve as template for the synthesis of catalase mimetics for therapeutic intervention in oxidative stress related disorders. However, unlike the heme catalases, structural aspects of Mn-catalases remain inadequately explored. The genome of the ancient cyanobacterium Anabaena PCC7120, shows the presence of two Mn-catalases, KatA and KatB. Here, we report the biochemical and structural characterization of KatB. The KatB protein (with a C-terminal his-tag) was over-expressed in Escherichia coli and purified by affinity chromatography. On the addition of Mn(2+) to the E. coli growth medium, a substantial increase in production of the soluble KatB protein was observed. The purified KatB protein was an efficient catalase, which was relatively insensitive to inhibition by azide. Crystal structure of KatB showed a hexameric assembly with four-helix bundle fold, characteristic of the Ferritin-like superfamily. With canonical Glu4His2 coordination geometry and two terminal water ligands, the KatB active site was distinctly different from that of other Mn-catalases. Interestingly, the KatB active site closely resembled the active sites of ruberythrin/bacterioferritin, bi-iron members of the Ferritin-like superfamily. The KatB crystal structure provided fundamental insights into the evolutionary relationship within the Ferritin-like superfamily and further showed that Mn-catalases can be sub-divided into two groups, each with a distinct active site configuration. Copyright © 2016 Elsevier Inc. All rights reserved.

A combinatorial feature selection approach to describe the QSAR of dual site inhibitors of acetylcholinesterase.

PubMed

Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz; Barkooie, Seyyed Mohsen Hosseini; Jahandideh, Samad; Rezaei, Mohammad Ali

2009-12-01

Regarding the great potential of dual binding site inhibitors of acetylcholinesterase as the future potent drugs of Alzheimer's disease, this study was devoted to extraction of the most effective structural features of these inhibitors from among a large number of quantitative descriptors. To do this, we adopted a unique approach in quantitative structure-activity relationships. An efficient feature selection method was emphasized in such an approach, using the confirmative results of different routine and novel feature selection methods. The proposed methods generated quite consistent results ensuring the effectiveness of the selected structural features.

Human prorenin structure sheds light on a novel mechanism of its autoinhibition and on its non-proteolytic activation by the (pro)renin receptor.

PubMed

Morales, Renaud; Watier, Yves; Böcskei, Zsolt

2012-08-03

Antibodies and prorenin mutants have long been used to structurally characterize prorenin, the inactive proenzyme form of renin. They were designed on the basis of homology models built using other aspartyl protease proenzyme structures since no structure was available for prorenin. Here, we present the first X-ray structure of a prorenin. The current structure of prorenin reveals that, in this zymogene, the active site of renin is blocked by the N-terminal residues of the mature version of the renin molecule, which are, in turn, covered by an Ω-shaped prosegment. This prevents access of substrates to the active site. The departure of the prosegment on activation induces an important global conformational change in the mature renin molecule with respect to prorenin: similar to other related enzymes such as pepsin or gastricsin, the segment that constitutes the N-terminal β-strand in renin is displaced from the renin active site by about 180° straight into the position that corresponds to the N-terminal β-strand of the prorenin prosegment. This way, the renin active site will become completely exposed and capable of carrying out its catalytic functions. A unique inactivation mechanism is also revealed, which does not make use of a lysine against the catalytic aspartates, probably in order to facilitate pH-independent activation [e.g., by the (pro)renin receptor]. Copyright © 2012 Elsevier Ltd. All rights reserved.

A comprehensive study of Superfund program benefits in the Denver and Tampa Bay metropolitan areas

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

Held, K.; Casper, B.; Siddhanti, S.K.

1995-12-31

The purpose of the study is to evaluate the benefits of the Superfund program in selected geographic areas. The study demonstrates how the cleanup of Superfund sites has improved the overall quality of life of those in the affected communities. The study presents findings on the benefits of Superfund cleanup activity in the Denver, Colorado and Tampa Bay, Florida metropolitan areas. Denver and Tampa Bay were chosen from several areas that the EPA evaluated and screened during the initial phase of the study. These locations were chosen because of a substantial presence of Superfund activities, making it possible to assessmore » the efficacy of the program. Several features make this study unique in terms of its overall goal. The study examines a broad range of benefit categories related to human health, environmental, and socioeconomic effects of Superfund cleanup activities. The study is also designed to assess benefits due to completed, current, and future planned activity at Superfund sites. This assessment covers Federal remedial activities at National Priorities List (NPL) sites, as well as relevant Federal removal actions in the study areas. These benefits are investigated from an area-wide perspective, as opposed to site-by-site, to determine Superfund`s overall effect on the communities in each area. The study consists of two major phases: Phase 1: Screening and ranking 16 prospective geographic areas and selecting Denver and Tampa Bay as the most appropriate areas for in-depth analysis; and Phase 2: Developing methodologies for assessing benefits, collecting relevant data, and analyzing the benefits from Superfund cleanup activity.« less

Teen smoking cessation help via the Internet: a survey of search engines.

PubMed

Edwards, Christine C; Elliott, Sean P; Conway, Terry L; Woodruff, Susan I

2003-07-01

The objective of this study was to assess Web sites related to teen smoking cessation on the Internet. Seven Internet search engines were searched using the keywords teen quit smoking. The top 20 hits from each search engine were reviewed and categorized. The keywords teen quit smoking produced between 35 and 400,000 hits depending on the search engine. Of 140 potential hits, 62% were active, unique sites; 85% were listed by only one search engine; and 40% focused on cessation. Findings suggest that legitimate on-line smoking cessation help for teens is constrained by search engine choice and the amount of time teens spend looking through potential sites. Resource listings should be updated regularly. Smoking cessation Web sites need to be picked up on multiple search engine searches. Further evaluation of smoking cessation Web sites need to be conducted to identify the most effective help for teens.

Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1

PubMed Central

Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y

2016-01-01

KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4+ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. DOI: http://dx.doi.org/10.7554/eLife.16093.001 PMID:27542194

Structure of the Arabidopsis Glucan Phosphatase LIKE SEX FOUR2 Reveals a Unique Mechanism for Starch Dephosphorylation[W

PubMed Central

Meekins, David A.; Guo, Hou-Fu; Husodo, Satrio; Paasch, Bradley C.; Bridges, Travis M.; Santelia, Diana; Kötting, Oliver; Vander Kooi, Craig W.; Gentry, Matthew S.

2013-01-01

Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase LIKE SEX FOUR2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules. PMID:23832589

Geoconservation - a southern African and African perspective

NASA Astrophysics Data System (ADS)

Reimold, Wolf Uwe

1999-10-01

In contrast to Europe, where geoconservation is actively pursued in most countries and where two international symposia on this subject have been staged in 1991 and 1996, geoconservation in Africa has indeed a very poor record. Considering the wealth of outstanding geological sites and the importance African stratigraphy has within the global geological record, pro-active geoconservation on this continent has not featured very prominently to date. In the interest of science, education and tourism, unique and typical geosites need to be identified, catalogued, and prioritised with the aim being their protection. Most African countries do not have vibrant non-governmental organisations such as a strong geological society, which could drive projects like geoconservation, or strong support from the private sector for environmental work. Here, a case is made for the role that established National Geological Surveys, some of which are already involved with retroactive environmental geological work, could play in the forefront of pro-active geoconservation and site protection.

Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors.

PubMed

Wang, Jingyi; Lindstrom, Jon

2018-06-01

Heteromeric nicotinic ACh receptors (nAChRs) were thought to have two orthodox agonist-binding sites at two α/β subunit interfaces. Highly selective ligands are hard to develop by targeting orthodox agonist sites because of high sequence similarity of this binding pocket among different subunits. Recently, unorthodox ACh-binding sites have been discovered at some α/α and β/α subunit interfaces, such as α4/α4, α5/α4 and β3/α4. Targeting unorthodox sites may yield subtype-selective ligands, such as those for (α4β2) 2 α5, (α4β2) 2 β3 and (α6β2) 2 β3 nAChRs. The unorthodox sites have unique pharmacology. Agonist binding at one unorthodox site is not sufficient to activate nAChRs, but it increases activation from the orthodox sites. NS9283, a selective agonist for the unorthodox α4/α4 site, was initially thought to be a positive allosteric modulator (PAM). NS9283 activates nAChRs with three engineered α4/α4 sites. PAMs, on the other hand, act at allosteric sites where ACh cannot bind. Known PAM sites include the ACh-homologous non-canonical site (e.g. morantel at β/α), the C-terminus (e.g. Br-PBTC and 17β-estradiol), a transmembrane domain (e.g. LY2087101) or extracellular and transmembrane domain interfaces (e.g. NS206). Some of these PAMs, such as Br-PBTC and 17β-estradiol, require only one subunit to potentiate activation of nAChRs. In this review, we will discuss differences between activation from orthosteric and allosteric sites, their selective ligands and clinical implications. These studies have advanced understanding of the structure, assembly and pharmacology of heteromeric neuronal nAChRs. This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc. © 2017 The British Pharmacological Society.

Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1

PubMed Central

Fay, Jonathan F.; Farrens, David L.

2015-01-01

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling. PMID:26100912

Co-Conserved MAPK Features Couple D-Domain Docking Groove to Distal Allosteric Sites via the C-Terminal Flanking Tail

PubMed Central

Nguyen, Tuan; Ruan, Zheng; Oruganty, Krishnadev; Kannan, Natarajan

2015-01-01

Mitogen activated protein kinases (MAPKs) form a closely related family of kinases that control critical pathways associated with cell growth and survival. Although MAPKs have been extensively characterized at the biochemical, cellular, and structural level, an integrated evolutionary understanding of how MAPKs differ from other closely related protein kinases is currently lacking. Here, we perform statistical sequence comparisons of MAPKs and related protein kinases to identify sequence and structural features associated with MAPK functional divergence. We show, for the first time, that virtually all MAPK-distinguishing sequence features, including an unappreciated short insert segment in the β4-β5 loop, physically couple distal functional sites in the kinase domain to the D-domain peptide docking groove via the C-terminal flanking tail (C-tail). The coupling mediated by MAPK-specific residues confers an allosteric regulatory mechanism unique to MAPKs. In particular, the regulatory αC-helix conformation is controlled by a MAPK-conserved salt bridge interaction between an arginine in the αC-helix and an acidic residue in the C-tail. The salt-bridge interaction is modulated in unique ways in individual sub-families to achieve regulatory specificity. Our study is consistent with a model in which the C-tail co-evolved with the D-domain docking site to allosterically control MAPK activity. Our study provides testable mechanistic hypotheses for biochemical characterization of MAPK-conserved residues and new avenues for the design of allosteric MAPK inhibitors. PMID:25799139

Binding Site Configurations Probe the Structure and Dynamics of the Zinc Finger of NEMO (NF-κB Essential Modulator).

PubMed

Godwin, Ryan C; Melvin, Ryan L; Gmeiner, William H; Salsbury, Freddie R

2017-01-31

Zinc-finger proteins are regulators of critical signaling pathways for various cellular functions, including apoptosis and oncogenesis. Here, we investigate how binding site protonation states and zinc coordination influence protein structure, dynamics, and ultimately function, as these pivotal regulatory proteins are increasingly important for protein engineering and therapeutic discovery. To better understand the thermodynamics and dynamics of the zinc finger of NEMO (NF-κB essential modulator), as well as the role of zinc, we present results of 20 μs molecular dynamics trajectories, 5 μs for each of four active site configurations. Consistent with experimental evidence, the zinc ion is essential for mechanical stabilization of the functional, folded conformation. Hydrogen bond motifs are unique for deprotonated configurations yet overlap in protonated cases. Correlated motions and principal component analysis corroborate the similarity of the protonated configurations and highlight unique relationships of the zinc-bound configuration. We hypothesize a potential mechanism for zinc binding from results of the thiol configurations. The deprotonated, zinc-bound configuration alone predominantly maintains its tertiary structure throughout all 5 μs and alludes rare conformations potentially important for (im)proper zinc-finger-related protein-protein or protein-DNA interactions.

Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe-S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity.

PubMed

Molle, Thibaut; Moreau, Yohann; Clemancey, Martin; Forouhar, Farhad; Ravanat, Jean-Luc; Duraffourg, Nicolas; Fourmond, Vincent; Latour, Jean-Marc; Gambarelli, Serge; Mulliez, Etienne; Atta, Mohamed

2016-10-18

RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C 3 methylthiolation of the D89 residue in the ribosomal S 12 protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO. Our data establish that the two SAM molecules bind the radical-SAM cluster to the unique iron site, and spectroscopic evidence obtained under strongly reducing conditions supports a mechanism in which the first molecule of SAM causes the reoxidation of the reduced radical-SAM cluster, impeding reductive cleavage of SAM to occur and allowing SAM to methylate a HS - ligand bound to the additional cluster. Furthermore, by using density functional theory-based methods, we provide a description of the reaction mechanism that predicts the attack of the carbon radical substrate on the methylthio group attached to the additional [4Fe-4S] cluster.

Distribution and Features of the Six Classes of Peroxiredoxins

PubMed Central

Poole, Leslie B.; Nelson, Kimberly J.

2016-01-01

Peroxiredoxins are cysteine-dependent peroxide reductases that group into 6 different, structurally discernable classes. In 2011, our research team reported the application of a bioinformatic approach called active site profiling to extract active site-proximal sequence segments from the 29 distinct, structurally-characterized peroxiredoxins available at the time. These extracted sequences were then used to create unique profiles for the six groups which were subsequently used to search GenBank(nr), allowing identification of ∼3500 peroxiredoxin sequences and their respective subgroups. Summarized in this minireview are the features and phylogenetic distributions of each of these peroxiredoxin subgroups; an example is also provided illustrating the use of the web accessible, searchable database known as PREX to identify subfamily-specific peroxiredoxin sequences for the organism Vitis vinifera (grape). PMID:26810075

Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

PubMed

Vatter, Heather A; Di, Han; Donaldson, Eric F; Radu, Gertrud U; Maines, Taronna R; Brinton, Margo A

2014-08-01

The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1β, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1β were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites for each PLP1 were confirmed by testing mutant constructs. Several unique features of the SHFV PLP1s were discovered. The SHFV PLP1α catalytic Cys63 is unique among arterivirus PLP1s in being adjacent to an Ala instead of a Trp. Other arterivirus PLP1s cleave only in cis at a single downstream site, but SHFV PLP1γ can cleave at both the downstream nsp1γ-nsp2 and upstream nsp1β-nsp1γ junctions. The three mature nsp1 proteins were produced both in the in vitro reactions and in infected cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A

PubMed Central

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

Crystal Structure of Cockroach Allergen Bla g 2, an Unusual Zinc Binding Aspartic Protease with a Novel Mode of Self-inhibition

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

Gustchina, Alla; Li, Mi; Wunschmann, Sabina

2010-07-19

The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity ofmore » the catalytic aspartate residues, increasing the distance between them to {approx}4 {angstrom} and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.« less

Decaleside: a new class of natural insecticide targeting tarsal gustatory sites

NASA Astrophysics Data System (ADS)

Rajashekar, Yallappa; Rao, Lingamallu J. M.; Shivanandappa, Thimmappa

2012-10-01

Natural sources for novel insecticide molecules hold promise in view of their eco-friendly nature, selectivity, and mammalian safety. Recent progress in understanding the biology of insect olfaction and taste offers new strategies for developing selective pest control agents. We have isolated two natural insecticidal molecules from edible roots of Decalepis hamiltonii named Decalesides I and II, which are novel trisaccharides, highly toxic to household insect pests and stored-product insects. We have experimentally shown that insecticidal activity requires contact with tarsi on the legs but is not toxic orally. The insecticidal activity of molecules is lost by hydrolysis, and various sugars modify toxic response, showing that the insecticidal activity is via gustatory sites on the tarsi. Selective toxicity to insects by virtue of their gustatory site of action and the mammalian safety of the new insecticides is inherent in their chemical structure with 1-4 or 1-1 α linkage that is easily hydrolyzed by digestive enzymes of mammals. Decalesides represent a new chemical class of natural insecticides with a unique mode of action targeting tarsal chemosensory/gustatory system of insects.

A designed inhibitor of a CLC antiporter blocks function through a unique binding mode

PubMed Central

Howery, Andrew E.; Elvington, Shelley; Abraham, Sherwin J.; Choi, Kee-Hyun; Phillips, Sabrina; Ryan, Christopher M.; Sanford, R. Lea; Simpson-Dworschak, Sierra; Almqvist, Jonas; Tran, Kevin; Chew, Thomas A.; Zachariae, Ulrich; Andersen, Olaf S.; Whitelegge, Julian; Matulef, Kimberly; Du Bois, Justin; Maduke, Merritt C.

2012-01-01

SUMMARY The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC “Chloride Channel” family represents a unique target for biophysical and biochemical studies because its distinctive protein fold supports both passive chloride channels and secondary-active chloride-proton transporters. Here, we describe the synthesis and characterization of the first specific small-molecule inhibitor directed against a CLC antiporter (ClC-ec1). This compound, 4,4′-octanamidostilbene-2,2′-disulfonate (OADS), inhibits ClC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (ClC-1). Inhibition of ClC-ec1 occurs by binding to two distinct intracellular sites. The location of these sites and the lipid-dependence of inhibition suggest potential mechanisms of action. The discovery of this compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders. PMID:23177200

Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

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

Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.

Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolutionmore » X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.« less

Chloride sensing by WNK1 kinase involves inhibition of autophosphorylation

PubMed Central

Piala, Alexander T.; Moon, Thomas M.; Akella, Radha; He, Haixia; Cobb, Melanie H.; Goldsmith, Elizabeth J.

2014-01-01

WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation. PMID:24803536

There's Iron in Them Thar Hills: A Geologic Look at the Aristarchus Plateau as a Potential Landing Site for Human Lunar Return

NASA Technical Reports Server (NTRS)

Coombs, Cassandra R.; McKay, David S.

1997-01-01

Lunar pyroclastic deposits are unique among lunar soils. Composed of very fine grained glass beads rich in Fe, Ti and Mg they yield unique spectral signatures. From the spectra two major classes and five subclasses of lunar dark mantling deposits have been identified. Recent work by me and others has shown that the larger regional deposits are more numerous, extensive, thicker, and widely distributed than previously thought, leading us to suggest that they would make ideal resource feedstock for future lunar surface activities. Returned sample studies and the recently collected Galileo and Clementine data also corroborate these findings. Recent planning for return to the Moon indicates that large cost savings can result from using locally produced oxygen, and recent JSC laboratory results indicate that iron-rich pyroclastic dark mantling deposits may be the richest oxygen resource on the Moon. My earlier work demonstrated that instead of using regolith, bulk lunar pyroclastic deposits are better suited for beneficiation as they are thick (lO's m's), unconsolidated, fine-grained deposits. In addition, the lack of rocks and boulders and the typically flat to gently rolling terrain will facilitate their mining and processing. In preparation for the Human Lunar Return (HLR) I have characterized the Aristarchus Plateau (24 deg. N 52 deg. W) as a potential landing site for an in-situ resource utilization (ISRU) demonstration. The geologic diversity and large volume of Fe-rich pyroclastic material present at the Aristarchus site make it an ideal target for extracting O2, H2 and halogens. This paper (1) describes the current understanding of the geology of Aristarchus plateau; (2) describes the resource potential of the Aristarchus plateau; and (3) presents several candidate landing sites on the plateau for future lunar activities.

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

PubMed Central

Hennig, Sandra E.; Jeoung, Jae-Hun; Goetzl, Sebastian; Dobbek, Holger

2012-01-01

Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe4S4] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins. PMID:22431597

Bacterial communities and the nitrogen cycle in the gypsum soils of Cuatro Ciénegas Basin, coahuila: a Mars analogue.

PubMed

López-Lozano, Nguyen E; Eguiarte, Luis E; Bonilla-Rosso, Germán; García-Oliva, Felipe; Martínez-Piedragil, Celeste; Rooks, Christine; Souza, Valeria

2012-07-01

The OMEGA/Mars Express hyperspectral imager identified gypsum at several sites on Mars in 2005. These minerals constitute a direct record of past aqueous activity and are important with regard to the search of extraterrestrial life. Gale Crater was chosen as Mars Science Laboratory Curiosity's landing site because it is rich in gypsum, as are some desert soils of the Cuatro Ciénegas Basin (CCB) (Chihuahuan Desert, Mexico). The gypsum of the CCB, which is overlain by minimal carbonate deposits, was the product of magmatic activity that occurred under the Tethys Sea. To examine this Mars analogue, we retrieved gypsum-rich soil samples from two contrasting sites with different humidity in the CCB. To characterize the site, we obtained nutrient data and analyzed the genes related to the N cycle (nifH, nirS, and nirK) and the bacterial community composition by using 16S rRNA clone libraries. As expected, the soil content for almost all measured forms of carbon, nitrogen, and phosphorus were higher at the more humid site than at the drier site. What was unexpected is the presence of a rich and divergent community at both sites, with higher taxonomic diversity at the humid site and almost no taxonomic overlap. Our results suggest that the gypsum-rich soils of the CCB host a unique microbial ecosystem that includes novel microbial assemblies.

Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.

PubMed

Kjaergaard, Christian H; Qayyum, Munzarin F; Wong, Shaun D; Xu, Feng; Hemsworth, Glyn R; Walton, Daniel J; Young, Nigel A; Davies, Gideon J; Walton, Paul H; Johansen, Katja Salomon; Hodgson, Keith O; Hedman, Britt; Solomon, Edward I

2014-06-17

Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.

Voluntary switching between identities in dissociative identity disorder: A functional MRI case study.

PubMed

Savoy, R L; Frederick, B B; Keuroghlian, A S; Wolk, P C

2012-01-01

Patients who suffer from dissociative identity disorder present unique scientific and clinical challenges for psychology and psychiatry. We have been fortunate in working with a patient who-while undergoing functional MRI-can switch rapidly and voluntarily between her main personality (a middle-aged, high-functioning woman) and an alternate personality (a 4-6-year-old girl). A unique task was designed to isolate the processes occurring during the switches between these personalities. Data are from two imaging sessions, conducted months apart, each showing the same activated areas during switches between these personalities. The activated areas include the following: the primary sensory and motor cortex, likely associated with characteristic facial movements made during switching; the nucleus accumbens bilaterally, possibly associated with aspects of reward connected with switching; and prefrontal sites, presumably associated with the executive control involved in the switching of personalities.

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

Mehboob, Shahila; Hevener, Kirk E.; Truong, Kent

Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motifmore » of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.« less

How technology transfer issues are managed

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

Sink, C.H.; Easley, K.R.

1991-12-31

In 1989, Secretary of Energy James Watkins made a commitment to accelerate DOE compliance with all applicable laws and standards aimed at protecting human health and the environment. At a minimum, this pledge requires the remediation of the 1989 inventory of chemical, radioactive, and mixed wastes at DOE production sites by 2019. The 1989 Complex inventory consisted of more than 3,700 sites, encompassing more than 26,000 acres contaminated with radioactive, hazardous, and mixed wastes. In addition, over 500 surplus sites are awaiting decontamination and decommissioning (D and D), and approximately 5,000 peripheral properties have contaminated soils (e.g., uranium tailings). Moreover,more » these problems exist at both inactive sites, where the primary focus is on environmental restoration, and at active sites, where the major emphasis is on improved waste management techniques. Although some of DOE`s problems are considered unique due to radioactivity, most forms of contamination resident in the Complex are not; rather, contaminants such as waste chemicals (e.g., inorganics), organics (e.g., fuels and solvents), halogenated organics (e.g., PCBs) and heavy metals commonly result in conventional industrial processes. Although certain other forms of contamination are more unique to DOE operations (e.g., radioactive materials, explosives, and pyrophorics), they are not exclusive to DOE. As DOE develops innovative solutions to these and related waste problems, it is imperative that technology systems and lessons learned be transferred from DOE sites and its R and D laboratories to private industry to maximize the nation`s return on environmental management technology investments.« less

Theoretical Investigations into Defected Graphene for Electrochemical Reduction of CO 2

DOE PAGES

Siahrostami, Samira; Jiang, Kun; Karamad, Mohammadreza; ...

2017-10-10

Here, despite numerous experimental efforts that have been dedicated to studying carbon-based materials for electrochemical reduction of CO 2, a rationalization of the associated trends in the intrinsic activity of different active motifs has so far been elusive. In the present work, we employ density functional theory calculations to examine a variety of different active sites in N-doped graphene to give a comprehensive outline of the trends in activity. We find that adsorption energies of COOH* and CO* do not follow the linear scaling relationships observed for the pure transition metals, and this unique scaling is rationalized through differences inmore » electronic structure between transition metals and defected graphene. This finding rationalizes most of the experimental observations on the carbon-based materials which present promising catalysts for the two-electron reduction of CO 2 to CO. With this simple thermodynamic analysis, we identify several active sites that are expected to exhibit a comparable or even better activity to the state-of-the-art gold catalyst, and several configurations are suggested to be selective for CO 2RR over HER.« less

Towards a mechanistic and physiological understanding of a ferredoxin disulfide reductase from the domains Archaea and Bacteria.

PubMed

Prakash, Divya; Walters, Karim A; Martinie, Ryan J; McCarver, Addison C; Kumar, Adepu K; Lessner, Daniel J; Krebs, Carsten; Golbeck, John H; Ferry, James G

2018-05-02

Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster.  UV-Vis, EPR and Mӧssbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]1+ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active-site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR advancing the physiological understanding of FDRs role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show FDR homologs are widespread in diverse microbes from the domain Bacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Novel synthesis and structural characterization of a high-affinity paramagnetic kinase probe for the identification of non-ATP site binders by nuclear magnetic resonance.

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

Moy, Franklin J.; Lee, Arthur; Gavrin, Lori Krim

2010-07-23

To aid in the pursuit of selective kinase inhibitors, we have developed a unique ATP site binder tool for the detection of binders outside the ATP site by nuclear magnetic resonance (NMR). We report here the novel synthesis that led to this paramagnetic spin-labeled pyrazolopyrimidine probe (1), which exhibits nanomolar inhibitory activity against multiple kinases. We demonstrate the application of this probe by performing NMR binding experiments with Lck and Src kinases and utilize it to detect the binding of two compounds proximal to the ATP site. The complex structure of the probe with Lck is also presented, revealing howmore » the probe fits in the ATP site and the specific interactions it has with the protein. We believe that this spin-labeled probe is a valuable tool that holds broad applicability in a screen for non-ATP site binders.« less

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

PubMed

Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

2015-10-01

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

Structural modeling of glucanase-substrate complexes suggests a conserved tyrosine is involved in carbohydrate recognition in plant 1,3-1,4-β- d-glucanases

NASA Astrophysics Data System (ADS)

Tsai, Li-Chu; Chen, Yi-Ning; Shyur, Lie-Fen

2008-12-01

Glycosyl hydrolase family 16 (GHF16) truncated Fibrobacter succinogenes (TFs) and GHF17 barley 1,3-1,4-β- d-glucanases (β-glucanases) possess different structural folds, β-jellyroll and (β/α)8, although they both catalyze the specific hydrolysis of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in mixed β-1,3 and β-1,4 β- d-glucans or lichenan. Differences in the active site region residues of TFs β-glucanase and barley β-glucanase create binding site topographies that require different substrate conformations. In contrast to barley β-glucanase, TFs β-glucanase possesses a unique and compact active site. The structural analysis results suggest that the tyrosine residue, which is conserved in all known 1,3-1,4-β- d-glucanases, is involved in the recognition of mixed β-1,3 and β-1,4 linked polysaccharide.

Pt-Pd Bimetal Popcorn Nanocrystals: Enhancing the Catalytic Performance by Combination Effect of Stable Multipetals Nanostructure and Highly Accessible Active Sites.

PubMed

Ma, Yanxia; Yin, Lisi; Cao, Guojian; Huang, Qingli; He, Maoshuai; Wei, Wenxian; Zhao, Hong; Zhang, Dongen; Wang, Mingyan; Yang, Tao

2018-04-01

Exploration of highly efficient electrocatalysts is significantly urgent for the extensive adoption of the fuel cells. Because of their high activity and super stability, Pt-Pd bimetal nanocrystals have been widely recognized as one class of promising electrocatalysts for oxygen reduction. This article presents the synthesis of popcorn-shaped Pt-Pd bimetal nanoparticles with a wide composition range through a facile hydrothermal strategy. The hollow-centered nanoparticles are surrounded by several petals and concave surfaces. By exploring the oxygen reduction reaction on the carbon supported Pt-Pd popcorns in perchloric acid solution, it is found that compared with the commercial Pt/C catalyst the present catalysts display superior catalytic performances in aspects of catalytic activity and stability. More importantly, the Pt-Pd popcorns display minor performance degradations through prolonged potential cycling. The enhanced performances can be mainly attributed to the unique popcorn structure of the Pt-Pd components, which allows the appearance and long existence of the high active sites with more accessibility. The present work highlights the key roles of accessible high active sites in the oxygen reduction reaction, which will ultimately guide the design of highly durable Pt-Pd catalysts. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In Search of the Active Site of PMMO Enzyme: Partnership between a K-12 Teacher, a Graduate K-12 Teaching Fellow, and a Research Mentor

ERIC Educational Resources Information Center

Bearden, Katherine K.; Mainardi, Daniela S.; Culligan, Tanya

2009-01-01

The partnership between a K-12 teacher (Culligan), an NSF GK-12 Teaching Fellow graduate student (Bearden), and a Louisiana Tech faculty member (Mainardi) collaborating in a research and education project is described in this work. The unique grouping of these three researchers allows for maximum dissemination of developed modules. By the end of…

Magnetic exploration of a low-temperature ultramafic-hosted hydrothermal site (Lost City, 30°N, MAR)

NASA Astrophysics Data System (ADS)

Szitkar, Florent; Tivey, Maurice A.; Kelley, Deborah S.; Karson, Jeffrey A.; Früh-Green, Gretchen L.; Denny, Alden R.

2017-03-01

A 2003 high-resolution magnetic survey conducted by the Autonomous Underwater Vehicle ABE over the low-temperature, ultramafic-hosted hydrothermal field Lost City reveals a weak positive magnetic anomaly. This observation is in direct contrast to recent observations of strong positive magnetic anomalies documented over the high-temperature ultramafic-hosted hydrothermal vents fields Rainbow and Ashadze, which indicates that temperature may control the production of magnetization at these sites. The Lost City survey provides a unique opportunity to study a field that is, to date, one of a kind, and is an end member of ultramafic-hosted hydrothermal systems. Our results highlight the key contribution of temperature on magnetite production resulting from serpentinization reactions. Whereas high temperature promotes significant production and partitioning of iron into magnetite, low temperature favors iron partitioning into various alteration phases, resulting in a magnetite-poor rock. Moreover, the distribution of magnetic anomalies confirms results of a previous geological survey indicating the progressive migration of hydrothermal activity upslope. These discoveries contribute to the results of 25 yrs of magnetic exploration of a wide range of hydrothermal sites, from low- to high-temperature and from basalt- to ultramafic-hosted, and thereby validate using high-resolution magnetics as a crucial parameter for locating and characterizing hydrothermal sites hosting unique chemosynthetic-based ecosystems and potentially mineral-rich deposits.

Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

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

Shi, Rongxin; Mullins, Elwood A.; Shen, Xing‐Xing

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct frommore » that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.« less

Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

PubMed

Miura, Hiroshi; Mogi, Tatsushi; Ano, Yoshitaka; Migita, Catharina T; Matsutani, Minenosuke; Yakushi, Toshiharu; Kita, Kiyoshi; Matsushita, Kazunobu

2013-06-01

Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

Estrogen and androgen receptor activities of hydraulic fracturing chemicals and surface and ground water in a drilling-dense region

USGS Publications Warehouse

Kassotis, Christopher D.; Tillitt, Donald E.; Davis, J. Wade; Hormann, Anette M.; Nagel, Susan C.

2014-01-01

The rapid rise in natural gas extraction using hydraulic fracturing increases the potential for contamination of surface and ground water from chemicals used throughout the process. Hundreds of products containing more than 750 chemicals and components are potentially used throughout the extraction process, including more than 100 known or suspected endocrine-disrupting chemicals. We hypothesized thataselected subset of chemicalsusedin natural gas drilling operationsandalso surface and ground water samples collected in a drilling-dense region of Garfield County, Colorado, would exhibit estrogen and androgen receptor activities. Water samples were collected, solid-phase extracted, and measured for estrogen and androgen receptor activities using reporter gene assays in human cell lines. Of the 39 unique water samples, 89%, 41%, 12%, and 46% exhibited estrogenic, antiestrogenic, androgenic, and antiandrogenic activities, respectively. Testing of a subset of natural gas drilling chemicals revealed novel antiestrogenic, novel antiandrogenic, and limited estrogenic activities. The Colorado River, the drainage basin for this region, exhibited moderate levels of estrogenic, antiestrogenic, and antiandrogenic activities, suggesting that higher localized activity at sites with known natural gas–related spills surrounding the river might be contributing to the multiple receptor activities observed in this water source. The majority of water samples collected from sites in a drilling-dense region of Colorado exhibited more estrogenic, antiestrogenic, or antiandrogenic activities than reference sites with limited nearby drilling operations. Our data suggest that natural gas drilling operationsmayresult in elevated endocrine-disrupting chemical activity in surface and ground water.

Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodomys merriami) exposed to radionuclide contamination at the Nevada Test Site, USA

USGS Publications Warehouse

Theodorakis, Christopher W.; Bickham, John W.; Lamb, Trip; Medica, Philip A.; Lyne, T. Barrett

2001-01-01

We examined effects of radionuclide exposure at two atomic blast sites on kangaroo rats (Dipodomys merriami) at the Nevada Test Site, Nevada, USA, using genotoxicity and population genetic analyses. We assessed chromosome damage by micronucleus and flow cytometric assays and genetic variation by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (mtDNA) analyses. The RAPD analysis showed no population structure, but mtDNA exhibited differentiation among and within populations. Genotoxicity effects were not observed when all individuals were analyzed. However, individuals with mtDNA haplotypes unique to the contaminated sites had greater chromosomal damage than contaminated-site individuals with haplotypes shared with reference sites. When interpopulation comparisons used individuals with unique haplotypes, one contaminated site had greater levels of chromosome damage than one or both of the reference sites. We hypothesize that shared-haplotype individuals are potential migrants and that unique-haplotype individuals are potential long-term residents. A parsimony approach was used to estimate the minimum number of migration events necessary to explain the haplotype distributions on a phylogenetic tree. The observed predominance of migration events into the contaminated sites supported our migration hypothesis. We conclude the atomic blast sites are ecological sinks and that immigration masks the genotoxic effects of radiation on the resident populations.

PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal

PubMed Central

Drees, Steffen Lorenz; Li, Chan; Prasetya, Fajar; Saleem, Muhammad; Dreveny, Ingrid; Williams, Paul; Hennecke, Ulrich; Emsley, Jonas; Fetzner, Susanne

2016-01-01

Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis. PMID:26811339

Functional Studies on Oligotropha carboxidovorans Molybdenum-Copper CO Dehydrogenase Produced in Escherichia coli.

PubMed

Kaufmann, Paul; Duffus, Benjamin R; Teutloff, Christian; Leimkühler, Silke

2018-04-30

The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO 2 and the oxidation of H 2 to protons and electrons. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H 2 oxidation of Mo/Cu-dependent CODHs in the future.

Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

PubMed

Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

1995-02-10

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

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

Serer, María I.; Bonomi, Hernán R.; Guimarães, Beatriz G.

This work reports crystal structures of trimeric riboflavin synthase from the pathogen B. abortus both as the apo protein and in complex with several ligands of interest. It is shown that ligand binding drives the assembly of the unique active site of the trimer, and these findings are complemented by a detailed kinetic study on this enzyme, in which marked inhibition by substrate and product was observed. Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one moleculemore » of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C{sub 3} symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.« less

Catalytic activity of certain antibodies as a potential tool for drug synthesis and for directed prodrug therapies.

PubMed

Wójcik, T; Kieć-Kononowicz, K

2008-01-01

Catalytic activity of certain antibodies was proposed by Linus Pauling for the very first time more than six decades ago. Since then few examples of catalytic antibodies (abzymes) were found in human organism. From late 80's many synthetic abzymes were obtained after immunization by Transition State Analogs (TSA). Another approach is based on functional mimicry of antibody to an active site of an enzyme. Detection of an abzymatic activity requires special immunoassays. This unique strategy can be employed for new methods of drug synthesis, as well as for in vivo therapies. Catalytic antibodies seem to be a promising tool for therapeutic purposes, because of their specifity and stereoselectivity.

The Crystal Structure Analysis of Group B Streptococcus Sortase C1: A Model for the ;Lid; Movement upon Substrate Binding

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

Khare, Baldeep; Fu, Zheng-Qing; Huang, I-Hsiu

2012-02-07

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible 'lid' anchored in the active site. However, the mechanistic details of the 'lid' displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its 'lid' mutant and proposed a role of the 'lid' as a protectormore » of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.« less

High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with α-l-arabinofuranosidase activity

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

Hassan, Noor; Karolinska Institutet, Stockholm; Kori, Lokesh D.

2015-02-19

The crystal structure of the H. orenii glycosidase was determined by molecular replacement and refined at 1.10 Å resolution. A gene from the heterotrophic, halothermophilic marine bacterium Halothermothrix orenii has been cloned and overexpressed in Escherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced by H. orenii. The crystal structure of the H. orenii glycosidase was determined by molecular replacement and refined at 1.10 Å resolution. As for other GH43 members, the enzyme folds as a five-bladed β-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermalmore » denaturation data, the H. orenii glycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4 M NaCl and Mn{sup 2+} at pH 6.5. The gene encoding the H. orenii GH43 enzyme has previously been annotated as a putative α-l-arabinofuranosidase. Activity was detected with p-nitrophenyl-α-l-arabinofuranoside as a substrate, and therefore the name HoAraf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4 M NaCl and Mn{sup 2+} at pH 6.5, giving a specific activity of 20–36 µmol min{sup −1} mg{sup −1}. The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution of HoAraf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.« less

C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells.

PubMed

Fujiwara, Miho; Kato, Shintaro; Niwa, Yuki; Suzuki, Takehiro; Tsuchiya, Miyu; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

2016-08-01

R-spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β-catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C-mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C-mannosylation sites at Trp(153) and Trp(156) ; however, it had been unclear whether these sites are C-mannosylated or not. We demonstrated that Rspo3 was C-mannosylated at both Trp(153) and Trp(156) by mass spectrometry. Using C-mannosylation-defective Rspo3 mutant-overexpressing cell lines, we found that C-mannosylation of Rspo3 promotes its secretion and activates Wnt/β-catenin signaling. © 2016 Federation of European Biochemical Societies.

Automated Rocket Propulsion Test Management

NASA Technical Reports Server (NTRS)

Walters, Ian; Nelson, Cheryl; Jones, Helene

2007-01-01

The Rocket Propulsion Test-Automated Management System provides a central location for managing activities associated with Rocket Propulsion Test Management Board, National Rocket Propulsion Test Alliance, and the Senior Steering Group business management activities. A set of authorized users, both on-site and off-site with regard to Stennis Space Center (SSC), can access the system through a Web interface. Web-based forms are used for user input with generation and electronic distribution of reports easily accessible. Major functions managed by this software include meeting agenda management, meeting minutes, action requests, action items, directives, and recommendations. Additional functions include electronic review, approval, and signatures. A repository/library of documents is available for users, and all items are tracked in the system by unique identification numbers and status (open, closed, percent complete, etc.). The system also provides queries and version control for input of all items.

The unique role of the visual word form area in reading.

PubMed

Dehaene, Stanislas; Cohen, Laurent

2011-06-01

Reading systematically activates the left lateral occipitotemporal sulcus, at a site known as the visual word form area (VWFA). This site is reproducible across individuals/scripts, attuned to reading-specific processes, and partially selective for written strings relative to other categories such as line drawings. Lesions affecting the VWFA cause pure alexia, a selective deficit in word recognition. These findings must be reconciled with the fact that human genome evolution cannot have been influenced by such a recent and culturally variable activity as reading. Capitalizing on recent functional magnetic resonance imaging experiments, we provide strong corroborating evidence for the hypothesis that reading acquisition partially recycles a cortical territory evolved for object and face recognition, the prior properties of which influenced the form of writing systems. Copyright © 2011 Elsevier Ltd. All rights reserved.

Application of individual foraminifera Mg/Ca and δ18O analyses for paleoceanographic reconstructions in the Bay of Bengal and other active depositional environments

NASA Astrophysics Data System (ADS)

Fritz-Endres, T.; Dekens, P.; Fehrenbacher, J. S.; Spero, H. J.; Stine, A.

2017-12-01

Paleoceanographic research traditionally focuses on regions where sediment deposition is minimally affected by transport. However, sediment fans near tectonically active regions provide an opportunity to link oceanographic climate to terrestrial processes. Sediment cores recovered during IODP Expedition 354 in the Bay of Bengal include hemipelagic sections that record the history of tectonic uplift and the development of the Indian Monsoon through the last 10 Ma. Although these cores provide a unique opportunity to link marine and terrestrial climate, the complex depositional environment requires that the source of foraminifera is carefully considered before using these proxies to reconstruct oceanographic conditions. Foraminifera in Bengal Fan sediments may have been transported via turbidity currents from the northern Bay of Bengal, where the seasonal variability of SST and SSS is larger compared to the southern Bay of Bengal. We measured single Globigerinoides sacculifer Mg/Ca and δ18O from mudline samples of IODP Site U1454 (8.4°N, 85.5°E, 3721 m water depth) near the modern active channel and Site U1449 (8.4°N, 88.7°E, 3653 m water depth) far from channel activity. We compare these sites to single G. sacculifer from the core-top sample of Site 342KL (20.6°N, 90.1°E, 1256 m water depth) located on the continental shelf. Each foraminifera lives 2-4 weeks and the distribution of 60 to 80 data points reflects the seasonal range of SST and SSS at the location where the foraminifera calcified. Measurements in foraminifera from Site U1449 (away from active channel) are statistically different from the site in the northern Bay of Bengal and more consistent with local conditions. Conversely, foraminifera from the site near the active channel reflect a combined signal of local conditions recorded from the site far from channel activity and those recorded from the continental shelf. This suggests a portion of foraminifera from the active channel site have been transported from the northern Bay of Bengal. Our data show that foraminifera can be used to reconstruct SST and δ18O in this complex depositional environment, but caution must be taken when the down-core lithology indicates turbidites and possible sediment transport

The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

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

Bergamin, E.; Hallock, P; Burden, S

Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK.more » The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.« less

The herpesvirus proteases as targets for antiviral chemotherapy.

PubMed

Waxman, L; Darke, P L

2000-01-01

Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure-activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

PubMed

Farb, Joshua N; Morrical, Scott W

2009-01-16

Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

Methane Oxidation on Pd-Ceria. A DFT Study of the Combustion Mechanism over Pd, PdO and Pd-ceria Sites

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

Mayernick, Adam D.; Janik, Michael J.

2010-12-24

Palladium/ceria exhibits unique catalytic activity for hydrocarbon oxidation; however, the chemical and structural properties of active sites on the palladium–ceria surface are difficult to characterize. Strong interactions between palladium and the ceria support stabilize oxidized Pd δ+ species, which may contribute to the significant activity of Pd/ceria for methane oxidation. We present a density functional theory (DFT + U) investigation into methane oxidation over Pd/ceria and quantify the activity of the Pd xCe 1-xO 2(1 1 1) mixed oxide surface in comparison with the PdO(1 0 0) and Pd(1 1 1) surfaces. The methane activation barrier is lowest over themore » Pd xCe 1-xO 2(1 1 1) surface, even lower than over the Pd(1 1 1) surface or low coordinated stepped or kinked Pd sites. Subsequent reaction steps in complete oxidation, including product desorption and vacancy refilling, are considered to substantiate that methane activation remains the rate-limiting step despite the low barrier over Pd xCe 1-xO 2(1 1 1). The low barrier over the Pd xCe 1-xO 2(1 1 1) surface demonstrates that mixed ceria-noble metal oxides offer the potential for improved hydrocarbon oxidation performance with respect to dispersed noble metal particles on ceria.« less

Redox process is crucial for inhibitory properties of aurintricarboxylic acid against activity of YopH: virulence factor of Yersinia pestis

PubMed Central

Kuban-Jankowska, Alicja; Gorska, Magdalena; Tuszynski, Jack A; Ossowski, Tadeusz; Wozniak, Michal

2015-01-01

YopH is a bacterial protein tyrosine phosphatase, which is essential for the viability and pathogenic virulence of the plague-causing Yersinia sp. bacteria. Inactivation of YopH activity would lead to the loss of bacterial pathogenicity. We have studied the inhibitory properties of aurintricarboxylic acid (ATA) against YopH phosphatase and found that at nanomolar concentrations ATA reversibly decreases the activity of YopH. Computational docking studies indicated that in all binding poses ATA binds in the YopH active site. Molecular dynamics simulations showed that in the predicted binding pose, ATA binds to the essential Cys403 and Arg409 residues in the active site and has a stronger binding affinity than the natural substrate (pTyr). The cyclic voltammetry experiments suggest that ATA reacts remarkably strongly with molecular oxygen. Additionally, the electrochemical reduction of ATA in the presence of a negative potential from −2.0 to 2.5 V generates a current signal, which is observed for hydrogen peroxide. Here we showed that ATA indicates a unique mechanism of YopH inactivation due to a redox process. We proposed that the potent inhibitory properties of ATA are a result of its strong binding in the YopH active site and in situ generation of hydrogen peroxide near catalytic cysteine residue. PMID:26286963

Molecular phylogenetic and chemical analyses of the microbial mats in deep-sea cold seep sediments at the northeastern Japan Sea.

PubMed

Arakawa, Shizuka; Sato, Takako; Sato, Rumi; Zhang, Jing; Gamo, Toshitaka; Tsunogai, Urumu; Hirota, Akinari; Yoshida, Yasuhiko; Usami, Ron; Inagaki, Fumio; Kato, Chiaki

2006-08-01

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier ((13)C-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.

Astronomy Outreach for Large and Unique Audiences

NASA Astrophysics Data System (ADS)

Lubowich, D.; Sparks, R. T.; Pompea, S. M.; Kendall, J. S.; Dugan, C.

2013-04-01

In this session, we discuss different approaches to reaching large audiences. In addition to star parties and astronomy events, the audiences for some of the events include music concerts or festivals, sick children and their families, minority communities, American Indian reservations, and tourist sites such as the National Mall. The goal is to bring science directly to the public—to people who attend astronomy events and to people who do not come to star parties, science museums, or science festivals. These programs allow the entire community to participate in astronomy activities to enhance the public appreciation of science. These programs attract large enthusiastic crowds often with young children participating in these family learning experiences. The public will become more informed, educated, and inspired about astronomy and will also be provided with information that will allow them to continue to learn after this outreach activity. Large and unique audiences often have common problems, and their solutions and the lessons learned will be presented. Interaction with the participants in this session will provide important community feedback used to improve astronomy outreach for large and unique audiences. New ways to expand astronomy outreach to new large audiences will be discussed.

Mineralogical impact on long-term patterns of soil nitrogen and phosphorus enzyme activities

NASA Astrophysics Data System (ADS)

Mikutta, Robert; Turner, Stephanie; Meyer-Stüve, Sandra; Guggenberger, Georg; Dohrmann, Reiner; Schippers, Axel

2014-05-01

Soil chronosequences provide a unique opportunity to study microbial activity over time in mineralogical diverse soils of different ages. The main objective of this study was to test the effect of mineralogical properties, nutrient and organic matter availability over whole soil pro-files on the abundance and activity of the microbial communities. We focused on microbio-logical processes involved in nitrogen and phosphorus cycling at the 120,000-year Franz Josef soil chronosequence. Microbial abundances (microbial biomass and total cell counts) and enzyme activities (protease, urease, aminopeptidase, and phosphatase) were determined and related to nutrient contents and mineralogical soil properties. Both, microbial abundances and enzyme activities decreased with soil depth at all sites. In the organic layers, microbial biomass and the activities of N-hydrolyzing enzymes showed their maximum at the intermediate-aged sites, corresponding to a high aboveground biomass. In contrast, the phosphatase activity increased with site age. The activities of N-hydrolyzing enzymes were positively correlated with total carbon and nitrogen contents, whereas the phosphatase activity was negatively correlated with the phosphorus content. In the mineral soil, the enzyme activities were generally low, thus reflecting the presence of strongly sorbing minerals. Sub-strate-normalized enzyme activities correlated negatively to clay content as well as poorly crystalline Al and Fe oxyhydroxides, supporting the view that the evolution of reactive sec-ondary mineral phases alters the activity of the microbial communities by constraining sub-strate availability. Our data suggest a strong mineralogical influence on nutrient cycling par-ticularly in subsoil environments.

Technology Transfer of Biopolymer Soil Amendment for Rapid Revegetation and Erosion Control at Fort A. P. Hill, Virginia

DTIC Science & Technology

2016-05-01

consisting of a polysaccharide polymeric material, a natural product of plant/soil rhyzobial microbial activity, was demonstrated to enhance site...critical concern of the modern Army and the Army engineer. A unique soil additive consisting of a polysaccharide polymeric material, a natural product of... polysaccharide secreted by Rhizobium leguminosarum var. phaseoli CIAT 899. Carbohydrate Research 204: 103- 107. Kochian, L.V. 1995. Cellular mechanisms of

A NMDA receptor glycine site partial agonist, GLYX-13, simultaneously enhances LTP and reduces LTD at Schaffer collateral-CA1 synapses in hippocampus.

PubMed

Zhang, Xiao-lei; Sullivan, John A; Moskal, Joseph R; Stanton, Patric K

2008-12-01

N-methyl-D-aspartate glutamate receptors (NMDARs) are a key route for Ca2+ influx into neurons important to both activity-dependent synaptic plasticity and, when uncontrolled, triggering events that cause neuronal degeneration and death. Among regulatory binding sites on the NMDAR complex is a glycine binding site, distinct from the glutamate binding site, which must be co-activated for NMDAR channel opening. We developed a novel glycine site partial agonist, GLYX-13, which is both nootropic and neuroprotective in vivo. Here, we assessed the effects of GLYX-13 on long-term synaptic plasticity and NMDAR transmission at Schaffer collateral-CA1 synapses in hippocampal slices in vitro. GLYX-13 simultaneously enhanced the magnitude of long-term potentiation (LTP) of synaptic transmission, while reducing long-term depression (LTD). GLYX-13 reduced NMDA receptor-mediated synaptic currents in CA1 pyramidal neurons evoked by low frequency Schaffer collateral stimulation, but enhanced NMDAR currents during high frequency bursts of activity, and these actions were occluded by a saturating concentration of the glycine site agonist d-serine. Direct two-photon imaging of Schaffer collateral burst-evoked increases in [Ca2+] in individual dendritic spines revealed that GLYX-13 selectively enhanced burst-induced NMDAR-dependent spine Ca2+ influx. Examining the rate of MK-801 block of synaptic versus extrasynaptic NMDAR-gated channels revealed that GLYX-13 selectively enhanced activation of burst-driven extrasynaptic NMDARs, with an action that was blocked by the NR2B-selective NMDAR antagonist ifenprodil. Our data suggest that GLYX-13 may have unique therapeutic potential as a learning and memory enhancer because of its ability to simultaneously enhance LTP and suppress LTD.

Analogs of methyllycaconitine as novel noncompetitive inhibitors of nicotinic receptors: pharmacological characterization, computational modeling, and pharmacophore development.

PubMed

McKay, Dennis B; Chang, Cheng; González-Cestari, Tatiana F; McKay, Susan B; El-Hajj, Raed A; Bryant, Darrell L; Zhu, Michael X; Swaan, Peter W; Arason, Kristjan M; Pulipaka, Aravinda B; Orac, Crina M; Bergmeier, Stephen C

2007-05-01

As a novel approach to drug discovery involving neuronal nicotinic acetylcholine receptors (nAChRs), our laboratory targeted nonagonist binding sites (i.e., noncompetitive binding sites, negative allosteric binding sites) located on nAChRs. Cultured bovine adrenal cells were used as neuronal models to investigate interactions of 67 analogs of methyllycaconitine (MLA) on native alpha3beta4* nAChRs. The availability of large numbers of structurally related molecules presents a unique opportunity for the development of pharmacophore models for noncompetitive binding sites. Our MLA analogs inhibited nicotine-mediated functional activation of both native and recombinant alpha3beta4* nAChRs with a wide range of IC(50) values (0.9-115 microM). These analogs had little or no inhibitory effects on agonist binding to native or recombinant nAChRs, supporting noncompetitive inhibitory activity. Based on these data, two highly predictive 3D quantitative structure-activity relationship (comparative molecular field analysis and comparative molecular similarity index analysis) models were generated. These computational models were successfully validated and provided insights into the molecular interactions of MLA analogs with nAChRs. In addition, a pharmacophore model was constructed to analyze and visualize the binding requirements to the analog binding site. The pharmacophore model was subsequently applied to search structurally diverse molecular databases to prospectively identify novel inhibitors. The rapid identification of eight molecules from database mining and our successful demonstration of in vitro inhibitory activity support the utility of these computational models as novel tools for the efficient retrieval of inhibitors. These results demonstrate the effectiveness of computational modeling and pharmacophore development, which may lead to the identification of new therapeutic drugs that target novel sites on nAChRs.

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

Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.

DNA apurinic-apyrimidinic (AP) sites are prevalent noncoding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA-repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive, owing in part to limited structural information. Here we report multiple high-resolution human APE1-DNA structures that divulge new features of the APE1 reaction, including the metal-binding site, the nucleophile and the arginine clamps that mediate product release. We also report APE1-DNA structures with a T-G mismatch 5' to the AP site, representing a clustered lesion occurring in methylatedmore » CpG dinucleotides. Moreover, these structures reveal that APE1 molds the T-G mismatch into a unique Watson-Crick-like geometry that distorts the active site, thus reducing incision. Finally, these snapshots provide mechanistic clarity for APE1 while affording a rational framework to manipulate biological responses to DNA damage.« less

Capturing Snapshots of APE1 Processing DNA Damage

PubMed Central

Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; Dyrkheeva, Nadezhda S.; Wilson, Samuel H.

2015-01-01

DNA apurinic-apyrimidinic (AP) sites are prevalent non-coding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive due in part to limited structural information. We report multiple high-resolution human APE1:DNA structures that divulge novel features of the APE1 reaction, including the metal binding site, nucleophile, and arginine clamps that mediate product release. We also report APE1:DNA structures with a T:G mismatch 5′ to the AP-site, representing a clustered lesion occurring in methylated CpG dinucleotides. These reveal that APE1 molds the T:G mismatch into a unique Watson-Crick like geometry that distorts the active site reducing incision. These snapshots provide mechanistic clarity for APE1, while affording a rational framework to manipulate biological responses to DNA damage. PMID:26458045

Capturing snapshots of APE1 processing DNA damage

DOE PAGES

Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; ...

2015-10-12

DNA apurinic-apyrimidinic (AP) sites are prevalent noncoding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA-repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive, owing in part to limited structural information. Here we report multiple high-resolution human APE1-DNA structures that divulge new features of the APE1 reaction, including the metal-binding site, the nucleophile and the arginine clamps that mediate product release. We also report APE1-DNA structures with a T-G mismatch 5' to the AP site, representing a clustered lesion occurring in methylatedmore » CpG dinucleotides. Moreover, these structures reveal that APE1 molds the T-G mismatch into a unique Watson-Crick-like geometry that distorts the active site, thus reducing incision. Finally, these snapshots provide mechanistic clarity for APE1 while affording a rational framework to manipulate biological responses to DNA damage.« less

Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

NASA Astrophysics Data System (ADS)

Rosen, Christian B.; Kodal, Anne L. B.; Nielsen, Jesper S.; Schaffert, David H.; Scavenius, Carsten; Okholm, Anders H.; Voigt, Niels V.; Enghild, Jan J.; Kjems, Jørgen; Tørring, Thomas; Gothelf, Kurt V.

2014-09-01

DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

INS studies of Cobalt-Copper Catalyst for the Conversion of Syngas to Higher Oxygenates

NASA Astrophysics Data System (ADS)

Sprunger, Phillip; Wang, Zi; Patterson, Matthew; Kurtz, Richard; Spivey, James

Cobalt-copper catalysts have been proposed for the synthesis of ethanol and higher oxygenates as a substitute of Rh and other high-cost noble metal catalysts. Two types of sites with atomic proximity are needed to form higher oxygenates: one to dissociate CO and a second to insert CO to the intermediates to form the CHxCO intermediate. Metallic cobalt is responsible for CO dissociation, while the nature of the site for CO insertion is still under study. We have utilized inelastic neutron scattering (INS) at the VISION beamline at SNS to probe intermediate surface species of this cobalt-copper catalyst. This unique technique allows for elucidation of mechanistic details of the CO insertion and subsequent CHxCO intermediate formation on the metal surfaces (Co0, Co2C and/or Cu0) . In addition to XRD and EXAFS which show a unique surface Co-C carbide formation, a combination of both INS and computational modeling indicate that the active site for CHxCO intermediates. Sponsored through the Louisiana Consortium for Neutron Scattering, DOE No. DE-SC0012432 with additional support from the LA BOR; also ORNL's Spallation Neutron Source (VISION Beamline), DOE-BES under Contract No. DE-AC0500OR22725.

Implementing a user-driven online quality improvement toolkit for cancer care.

PubMed

Luck, Jeff; York, Laura S; Bowman, Candice; Gale, Randall C; Smith, Nina; Asch, Steven M

2015-05-01

Peer-to-peer collaboration within integrated health systems requires a mechanism for sharing quality improvement lessons. The Veterans Health Administration (VA) developed online compendia of tools linked to specific cancer quality indicators. We evaluated awareness and use of the toolkits, variation across facilities, impact of social marketing, and factors influencing toolkit use. A diffusion of innovations conceptual framework guided the collection of user activity data from the Toolkit Series SharePoint site and an online survey of potential Lung Cancer Care Toolkit users. The VA Toolkit Series site had 5,088 unique visitors in its first 22 months; 5% of users accounted for 40% of page views. Social marketing communications were correlated with site usage. Of survey respondents (n = 355), 54% had visited the site, of whom 24% downloaded at least one tool. Respondents' awareness of the lung cancer quality performance of their facility, and facility participation in quality improvement collaboratives, were positively associated with Toolkit Series site use. Facility-level lung cancer tool implementation varied widely across tool types. The VA Toolkit Series achieved widespread use and a high degree of user engagement, although use varied widely across facilities. The most active users were aware of and active in cancer care quality improvement. Toolkit use seemed to be reinforced by other quality improvement activities. A combination of user-driven tool creation and centralized toolkit development seemed to be effective for leveraging health information technology to spread disease-specific quality improvement tools within an integrated health care system. Copyright © 2015 by American Society of Clinical Oncology.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

PubMed Central

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase–an Essential Enzyme of the Polyamine Metabolism

PubMed Central

Krossa, Sebastian; Faust, Annette; Ober, Dietrich; Scheidig, Axel J.

2016-01-01

The highly conserved bacterial homospermidine synthase (HSS) is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila and Pseudomonas aeruginosa; The unique usage of NAD(H) as a prosthetic group is a common feature of bacterial HSS, eukaryotic HSS and deoxyhypusine synthase (DHS). The structure of the bacterial enzyme does not possess a lysine residue in the active center and thus does not form an enzyme-substrate Schiff base intermediate as observed for the DHS. In contrast to the DHS the active site is not formed by the interface of two subunits but resides within one subunit of the bacterial HSS. Crystal structures of Blastochloris viridis HSS (BvHSS) reveal two distinct substrate binding sites, one of which is highly specific for putrescine. BvHSS features a side pocket in the direct vicinity of the active site formed by conserved amino acids and a potential substrate discrimination, guiding, and sensing mechanism. The proposed reaction steps for the catalysis of BvHSS emphasize cation-π interaction through a conserved Trp residue as a key stabilizer of high energetic transition states. PMID:26776105

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

PubMed Central

Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

2013-01-01

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

PubMed

Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G

2013-12-27

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

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

PubMed

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

2013-04-05

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

The useful potential of using existing data to uniquely identify predictable wind events and regimes, part 1

NASA Technical Reports Server (NTRS)

Trettel, D. W.; Aquino, J. T.; Piazza, T. R.; Taylor, L. E.; Trask, D. C.

1982-01-01

Correlations between standard meteorological data and wind power generation potential were developed. Combined with appropriate wind forecasts, these correlations can be useful to load dispatchers to supplement conventional energy sources. Hourly wind data were analyzed for four sites, each exhibiting a unique physiography. These sites are Amarillo, Texas; Ludington, Michigan; Montauk Point, New York; and San Gorgonio, California. Synoptic weather maps and tables are presented to illustrate various wind 'regimes' at these sites.

Assessing the Deepwater Horizon oil spill impact on marine mammal population through acoustics: endangered sperm whales.

PubMed

Ackleh, Azmy S; Ioup, George E; Ioup, Juliette W; Ma, Baoling; Newcomb, Joal J; Pal, Nabendu; Sidorovskaia, Natalia A; Tiemann, Christopher

2012-03-01

Long-term monitoring of endangered species abundance based on acoustic recordings has not yet been pursued. This paper reports the first attempt to use multi-year passive acoustic data to study the impact of the Deepwater Horizon oil spill on the population of endangered sperm whales. Prior to the spill the Littoral Acoustic Demonstration Center (LADC) collected acoustic recordings near the spill site in 2007. These baseline data now provide a unique opportunity to better understand how the oil spill affected marine mammals in the Gulf of Mexico. In September 2010, LADC redeployed recording buoys at previously used locations 9, 25, and 50 miles away from the incident site. A statistical methodology that provides point and interval estimates of the abundance of the sperm whale population at the two nearest sites is presented. A comparison of the 2007 and the 2010 recordings shows a decrease in acoustic activity and abundance of sperm whales at the 9-mile site by a factor of 2, whereas acoustic activity and abundance at the 25-mile site has clearly increased. This indicates that some sperm whales may have relocated farther away from the spill. Follow-up experiments will be important for understanding long-term impact. © 2012 Acoustical Society of America

The neural signature of emotional memories in serial crimes.

PubMed

Chassy, Philippe

2017-10-01

Neural plasticity is the process whereby semantic information and emotional responses are stored in neural networks. It is hypothesized that the neural networks built over time to encode the sexual fantasies that motivate serial killers to act should display a unique, detectable activation pattern. The pathological neural watermark hypothesis posits that such networks comprise activation of brain sites that reflect four cognitive components: autobiographical memory, sexual arousal, aggression, and control over aggression. The neural sites performing these cognitive functions have been successfully identified by previous research. The key findings are reviewed to hypothesise the typical pattern of activity that serial killers should display. Through the integration of biological findings into one framework, the neural approach proposed in this paper is in stark contrast with the many theories accounting for serial killers that offer non-medical taxonomies. The pathological neural watermark hypothesis offers a new framework to understand and detect deviant individuals. The technical and legal issues are briefly discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase

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

Huerta, Carlos; Borek, Dominika; Machius, Mischa

2009-12-01

Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme flavin adenine dinucleotide (FAD) and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different sets of active-site residues to accomplish the same chemistry. Here we report the first structural characterization of a eukaryotic FMNAT from the pathogenic yeast Candida glabrata. Four crystal structures of C. glabrata FMNAT in different complexed forms were determined at 1.20-1.95 A resolutions, capturing the enzyme active-site states prior to and after catalysis. These structures reveal a novelmore » flavin-binding mode and a unique enzyme-bound FAD conformation. Comparison of the bacterial and eukaryotic FMNATs provides a structural basis for understanding the convergent evolution of the same FMNAT activity from different protein ancestors. Structure-based investigation of the kinetic properties of FMNAT should offer insights into the regulatory mechanisms of FAD homeostasis by FMNAT in eukaryotic organisms.« less

Engineering Ascorbate Peroxidase Activity Into Cytochrome C Peroxidase

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

Meharenna, Y.T.; Oertel, P.; Bhaskar, B.

2009-05-26

Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each others activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303--307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical argininemore » were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of {approx}12 min{sup -1}, indicating that the engineered ascorbate-binding loop can bind ascorbate.« less

Data-driven modeling of background and mine-related acidity and metals in river basins

USGS Publications Warehouse

Friedel, Michael J

2013-01-01

A novel application of self-organizing map (SOM) and multivariate statistical techniques is used to model the nonlinear interaction among basin mineral-resources, mining activity, and surface-water quality. First, the SOM is trained using sparse measurements from 228 sample sites in the Animas River Basin, Colorado. The model performance is validated by comparing stochastic predictions of basin-alteration assemblages and mining activity at 104 independent sites. The SOM correctly predicts (>98%) the predominant type of basin hydrothermal alteration and presence (or absence) of mining activity. Second, application of the Davies–Bouldin criteria to k-means clustering of SOM neurons identified ten unique environmental groups. Median statistics of these groups define a nonlinear water-quality response along the spatiotemporal hydrothermal alteration-mining gradient. These results reveal that it is possible to differentiate among the continuum between inputs of background and mine-related acidity and metals, and it provides a basis for future research and empirical model development.

Dynamic motif occupancy (DynaMO) analysis identifies transcription factors and their binding sites driving dynamic biological processes

PubMed Central

Kuang, Zheng; Ji, Zhicheng

2018-01-01

Abstract Biological processes are usually associated with genome-wide remodeling of transcription driven by transcription factors (TFs). Identifying key TFs and their spatiotemporal binding patterns are indispensable to understanding how dynamic processes are programmed. However, most methods are designed to predict TF binding sites only. We present a computational method, dynamic motif occupancy analysis (DynaMO), to infer important TFs and their spatiotemporal binding activities in dynamic biological processes using chromatin profiling data from multiple biological conditions such as time-course histone modification ChIP-seq data. In the first step, DynaMO predicts TF binding sites with a random forests approach. Next and uniquely, DynaMO infers dynamic TF binding activities at predicted binding sites using their local chromatin profiles from multiple biological conditions. Another landmark of DynaMO is to identify key TFs in a dynamic process using a clustering and enrichment analysis of dynamic TF binding patterns. Application of DynaMO to the yeast ultradian cycle, mouse circadian clock and human neural differentiation exhibits its accuracy and versatility. We anticipate DynaMO will be generally useful for elucidating transcriptional programs in dynamic processes. PMID:29325176

Neural systems underlying lexical retrieval for sign language.

PubMed

Emmorey, Karen; Grabowski, Thomas; McCullough, Stephen; Damasio, Hanna; Ponto, Laura L B; Hichwa, Richard D; Bellugi, Ursula

2003-01-01

Positron emission tomography was used to investigate whether signed languages exhibit the same neural organization for lexical retrieval within classical and non-classical language areas as has been described for spoken English. Ten deaf native American sign language (ASL) signers were shown pictures of unique entities (famous persons) and non-unique entities (animals) and were asked to name each stimulus with an overt signed response. Proper name signed responses to famous people were fingerspelled, and common noun responses to animals were both fingerspelled and signed with native ASL signs. In general, retrieving ASL signs activated neural sites similar to those activated by hearing subjects retrieving English words. Naming famous persons activated the left temporal pole (TP), whereas naming animals (whether fingerspelled or signed) activated left inferotemporal (IT) cortex. The retrieval of fingerspelled and native signs generally engaged the same cortical regions, but fingerspelled signs in addition activated a premotor region, perhaps due to the increased motor planning and sequencing demanded by fingerspelling. Native signs activated portions of the left supramarginal gyrus (SMG), an area previously implicated in the retrieval of phonological features of ASL signs. Overall, the findings indicate that similar neuroanatomical areas are involved in lexical retrieval for both signs and words. Copyright 2003 Elsevier Science Ltd.

The battle for hearts and minds: who is communicating most effectively with the cosmetic marketplace?

PubMed

Camp, Matthew C; Wong, Wendy W; Mussman, Jason L; Gupta, Subhas C

2010-01-01

Cosmetic surgery, historically the purview of plastic surgeons, has in recent years seen an influx of practitioners from other fields of training. Many of these new providers are savvy in marketing and public relations and are beginning to control a surprisingly large amount of cosmetic patient care. The purpose of this study is to measure the amount of traffic being attracted to the Web sites of individual practitioners and organizations vying for cosmetic patients. This study investigates the trends of the past 12 months and identifies changes of special concern to plastic surgeons. The Web sites of 1307 cosmetic providers were monitored over a year's time. The Web activity of two million individuals whose computers were loaded with a self-reporting software package was recorded and analyzed. The Web sites were analyzed according to the specialty training of the site owner and total unique visits per month were tallied for the most prominent specialties. The dominant Web sites were closely scrutinized and the Web optimization strategies of each were also examined. There is a tremendous amount of Web activity surrounding cosmetic procedures and the amount of traffic on the most popular sites is continuing to grow. Also, a large sum of money is being expended to channel Web traffic, with sums in the thousands of dollars being spent daily by top Web sites. Overall in the past year, the private Web sites of plastic surgeons have increased their reach by 10%, growing from 200,000 to approximately 220,000 unique visitors monthly. Plastic surgery remains the specialty with the largest number of Web visitors per month. However, when combined, the private Web sites of all other providers of aesthetic services have significantly outpaced plastic surgery's growth. The traffic going to non-plastic surgeons has grown by 50% (200,000 visitors per month in September 2008 to 300,000 visitors monthly in September 2009). For providers of aesthetic services, communication with the public is of utmost importance. The Web has become the single most important information resource for consumers because of easy access. Plastic surgeons are facing significant competition for the attention of potential patients, with increasingly sophisticated Web sites and listing services being set up by independent parties. It is important for plastic surgeons to become familiar with the available Internet tools for communication with potential patients and to aggressively utilize these tools for effective practice building.

Recognition of RNA Editing Sites Is Directed by Unique Proteins in Chloroplasts: Biochemical Identification of cis-Acting Elements and trans-Acting Factors Involved in RNA Editing in Tobacco and Pea Chloroplasts

PubMed Central

Miyamoto, Tetsuya; Obokata, Junichi; Sugiura, Masahiro

2002-01-01

RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants. PMID:12215530

In situ template synthesis of hollow nanospheres assembled from NiCo2S4@C ultrathin nanosheets with high electrochemical activities for lithium storage and ORR catalysis.

PubMed

Wu, Xiaoyu; Li, Songmei; Wang, Bo; Liu, Jianhua; Yu, Mei

2017-05-10

Transition-metal sulfide hollow nanostructures have received intensive attention in energy-related applications due to their unique structural features and high electrochemical activities. Here, a well-designed composite of NiCo 2 S 4 @C is successfully fabricated using a facile in situ template removal method. The obtained composite shows unique microstructures of hollow nanospheres (∼650 nm in diameter) assembled from ultrathin NiCo 2 S 4 @C nanosheets, in which numerous scattered NiCo 2 S 4 nanoparticles are embedded in ultrathin carbon nanosheets, exhibiting mesoporous features with a high surface area of 247.25 m 2 g -1 . When used as anode materials for LIBs, NiCo 2 S 4 @C hollow nanospheres exhibit a high reversible capacity of 1592 mA h g -1 at a current density of 500 mA g -1 , enhanced cycling performance maintaining a capacity of 1178 mA h g -1 after 200 cycles, and a remarkable rate capability. Meanwhile, the hollow nanospheres display excellent catalytic activity as ORR catalysts with a four-electron pathway and superior durability to that of commercial Pt/C catalysts. Their excellent lithium storage and ORR catalysis performance can be attributed to the rational incorporation of high-activity NiCo 2 S 4 and ultrathin carbon nanosheets, as well as unique hollow microstructures, which offer efficient electron/ion transport, an enhanced electroactive material/electrolyte contact area, numerous active sites, and excellent structural stability.

Greener synthesis of Cu-MOF-74 and its catalytic use for the generation of vanillin.

PubMed

Flores, J Gabriel; Sánchez-González, Elí; Gutiérrez-Alejandre, Aída; Aguilar-Pliego, Julia; Martínez, Ana; Jurado-Vázquez, Tamara; Lima, Enrique; González-Zamora, Eduardo; Díaz-García, Manuel; Sánchez-Sánchez, Manuel; Ibarra, Ilich A

2018-03-26

A greener synthesis of Cu-MOF-74 was obtained, for the first time, in methanol as the unique solvent and at room temperature. Full characterisation of the MOF material showed its purity and also its nanocrystalline nature. Complete activation (150 °C for 1 h and 10-3 bar) of Cu-MOF-74 afforded unsaturated Cu metal sites and this was corroborated by in situ DRIFT spectroscopy. The access to these Cu open metal sites was tested for the catalytic transformation of trans-ferulic acid to vanillin (yield of 71% and 97% selectivity) and a plausible catalytic reaction mechanism was postulated based on quantum chemical calculations.

Structure-Activity Relationship and Molecular Mechanics Reveal the Importance of Ring Entropy in the Biosynthesis and Activity of a Natural Product.

PubMed

Tran, Hai L; Lexa, Katrina W; Julien, Olivier; Young, Travis S; Walsh, Christopher T; Jacobson, Matthew P; Wells, James A

2017-02-22

Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues. To understand thiocillin's structure-activity relationship, we generated a site-saturation mutagenesis library covering each position along thiocillin's macrocyclic ring. We report the identification of eight unique compounds more potent than wild-type thiocillin, the best having an 8-fold improvement in potency. Computational modeling of thiocillin's macrocyclic structure revealed a striking requirement for a low-entropy macrocycle for activity. The populated ensembles of the active mutants showed a rigid structure with few adoptable conformations while inactive mutants showed a more flexible macrocycle which is unfavorable for binding. This finding highlights the importance of macrocyclization in combination with rigidifying post-translational modifications to achieve high-potency binding.

Inhibition of Monometalated Methionine Aminopeptidase: Inhibitor Discovery and Crystallographic Analysis†

PubMed Central

Huang, Min; Xie, Sheng-Xue; Ma, Ze-Qiang; Huang, Qing-Qing; Nan, Fa-Jun; Ye, Qi-Zhuang

2008-01-01

Two divalent metal ions are commonly seen in the active site cavity of methionine aminopeptidase, and at least one of the metal ions is directly involved in catalysis. Although ample structural and functional information is available for dimetalated enzyme, methionine aminopeptidase likely functions as a monometalated enzyme under physiological conditions. Information on structure, as well as catalysis and inhibition, of the monometalated enzyme is lacking. By improving conditions of high throughput screening, we identified a unique inhibitor with specificity toward the monometalated enzyme. Kinetic characterization indicates a mutual exclusivity in binding between the inhibitor and the second metal ion at the active site. This is confirmed by X-ray structure, and this inhibitor coordinates with the first metal ion and occupies the space normally occupied by the second metal ion. Kinetic and structural analyses of the inhibition by this and other inhibitors provide insight in designing effective inhibitors of methionine aminopeptidase. PMID:17948983

Catalase in peroxidase clothing: Interdependent cooperation of two cofactors in the catalytic versatility of KatG.

PubMed

Njuma, Olive J; Ndontsa, Elizabeth N; Goodwin, Douglas C

2014-02-15

Catalase-peroxidase (KatG) is found in eubacteria, archaea, and lower eukaryotae. The enzyme from Mycobacterium tuberculosis has received the greatest attention because of its role in activation of the antitubercular pro-drug isoniazid, and the high frequency with which drug resistance stems from mutations to the katG gene. Generally, the catalase activity of KatGs is striking. It rivals that of typical catalases, enzymes with which KatGs share no structural similarity. Instead, catalatic turnover is accomplished with an active site that bears a strong resemblance to a typical peroxidase (e.g., cytochrome c peroxidase). Yet, KatG is the only member of its superfamily with such capability. It does so using two mutually dependent cofactors: a heme and an entirely unique Met-Tyr-Trp (MYW) covalent adduct. Heme is required to generate the MYW cofactor. The MYW cofactor allows KatG to leverage heme intermediates toward a unique mechanism for H2O2 oxidation. This review evaluates the range of intermediates identified and their connection to the diverse catalytic processes KatG facilitates, including mechanisms of isoniazid activation. Copyright © 2013 Elsevier Inc. All rights reserved.

Preparation and Characterization of Mesoporous Nickel derived from Liquid crystalline Template and Evaluation of its Electro catalytic activity towards Methanol Oxidation

NASA Astrophysics Data System (ADS)

Mohanapriya, S.; Renuka devi, R.; Raj, V.

2018-02-01

Mesoporous Nickel has been prepared by electrodeposition using non-ionic surfactant based liquid crystalline template under optimized processing conditions. Physico-chemical properties of mesoporous nickel is systematically characterized through XRD, SEM and AFM analyses. Comparison of electrocatalytic activity of mesoporous nickel with smooth nickel was interrogated using cyclic voltammetry (CV), chronoamperometry (CA) and electrochemical impedance spectroscopy (EIS) analyses. Distinctly enhanced electrocatalytic activity with improved surface poisoning resistance related to mesoporous nickel electrode towards methanol oxidation stems from unique mesoporous morphology. This mesoporous morphology with high surface to volume ratio is highly beneficial to promote active catalytic centers to offer readily accessible Pt catalytic sites for MOR, through facilitating mass and electron transports.

Self-assembly of protein-based biomaterials initiated by titania nanotubes.

PubMed

Forstater, Jacob H; Kleinhammes, Alfred; Wu, Yue

2013-12-03

Protein-based biomaterials are a promising strategy for creating robust highly selective biocatalysts. The assembled biomaterials must sufficiently retain the near-native structure of proteins and provide molecular access to catalytically active sites. These requirements often exclude the use of conventional assembly techniques, which rely on covalent cross-linking of proteins or entrapment within a scaffold. Here we demonstrate that titania nanotubes can initiate and template the self-assembly of enzymes, such as ribonuclease A, while maintaining their catalytic activity. Initially, the enzymes form multilayer thick ellipsoidal aggregates centered on the nanotube surface; subsequently, these nanosized entities assemble into a micrometer-sized enzyme material that has enhanced enzymatic activity and contains as little as 0.1 wt % TiO2 nanotubes. This phenomenon is uniquely associated with the active anatase (001)-like surface of titania nanotubes and does not occur on other anatase nanomaterials, which contain significantly fewer undercoordinated Ti surface sites. These findings present a nanotechnology-enabled mechanism of biomaterial growth and open a new route for creating stable protein-based biomaterials and biocatalysts without the need for chemical modification.

Tissue-Specific Chromatin Modifications at a Multigene Locus Generate Asymmetric Transcriptional Interactions

PubMed Central

Yoo, Eung Jae; Cajiao, Isabela; Kim, Jeong-Seon; Kimura, Atsushi P.; Zhang, Aiwen; Cooke, Nancy E.; Liebhaber, Stephen A.

2006-01-01

Random assortment within mammalian genomes juxtaposes genes with distinct expression profiles. This organization, along with the prevalence of long-range regulatory controls, generates a potential for aberrant transcriptional interactions. The human CD79b/GH locus contains six tightly linked genes with three mutually exclusive tissue specificities and interdigitated control elements. One consequence of this compact organization is that the pituitarycell-specific transcriptional events that activate hGH-N also trigger ectopic activation of CD79b. However, the B-cell-specific events that activate CD79b do not trigger reciprocal activation of hGH-N. Here we utilized DNase I hypersensitive site mapping, chromatin immunoprecipitation, and transgenic models to explore the basis for this asymmetric relationship. The results reveal tissue-specific patterns of chromatin structures and transcriptional controls at the CD79b/GH locus in B cells distinct from those in the pituitary gland and placenta. These three unique transcriptional environments suggest a set of corresponding gene expression pathways and transcriptional interactions that are likely to be found juxtaposed at multiple sites within the eukaryotic genome. PMID:16847312

Azimuth selection for sea level measurements using geodetic GPS receivers

NASA Astrophysics Data System (ADS)

Wang, Xiaolei; Zhang, Qin; Zhang, Shuangcheng

2018-03-01

Based on analysis of Global Positioning System (GPS) multipath signals recorded by a geodetic GPS receiver, GPS Reflectometry (GPS-R) has demonstrated unique advantages in relation to sea level monitoring. Founded on multipath reflectometry theory, sea level changes can be measured by GPS-R through spectral analysis of recorded signal-to-noise ratio data. However, prior to estimating multipath parameters, it is necessary to define azimuth and elevation angle mask to ensure the reflecting zones are on water. Here, a method is presented to address azimuth selection, a topic currently under active development in the field of GPS-R. Data from three test sites: the Kachemak Bay GPS site PBAY in Alaska (USA), Friday Harbor GPS site SC02 in the San Juan Islands (USA), and Brest Harbor GPS site BRST in Brest (France) are analyzed. These sites are located in different multipath environments, from a rural coastal area to a busy harbor, and they experience different tidal ranges. Estimates by the GPS tide gauges at azimuths selected by the presented method are compared with measurements from physical tide gauges and acceptable correspondence found for all three sites.

Three-dimensional assembly structure of anatase TiO2 hollow microspheres with enhanced photocatalytic performance

NASA Astrophysics Data System (ADS)

Tang, Yihao; Zhan, Shuai; Wang, Li; Zhang, Bin; Ding, Minghui

The pure anatase TiO2 hollow microspheres are synthesized by a one-step template-free hydrothermal route. By defining temperature and time limits, we produce TiO2 hollow microspheres with a fluoride-mediated self-transformation. The surface morphology of TiO2 hollow microspheres was studied by SEM. The hollow microspheres have diameters of about 800 nm and are remarkably uniform. The UV-light photocatalytic activity and the stability/multifunction of TiO2 hollow microspheres structure were evaluated by photocatalytic degradation of methylene blue and photocatalytic hydrogen evolution. The excellent photocatalytic activity is attributed to large specific surface area, more active sites, unique hollow structures, and improved light scattering.

Structural and mechanistic insights into Mps1 kinase activation.

PubMed

Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

2009-08-01

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-A-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices EF and F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

The multiple nucleotide-divalent cation binding modes of Saccharomyces cerevisiae CK2α indicate a possible co-substrate hydrolysis product (ADP/GDP) release pathway.

PubMed

Liu, Huihui; Wang, Hong; Teng, Maikun; Li, Xu

2014-02-01

CK2 is a ubiquitous and conserved protein kinase in eukaryotic organisms and is important in many biological processes. It is unique in maintaining constitutive activity and in using both ATP and GTP as phosphor donors. In this study, crystal structures of recombinant Saccharomyces cerevisiae CK2α (scCK2α) complexed with GMPPNP, ATP and AMPPN with either Mg2+ or Mn2+ as the coordinated divalent cation are presented. The overall structure of scCK2α shows high similarity to its homologous proteins by consisting of two domains with the co-substrate lying in the cleft between them. However, three characteristic features distinguish scCK2α from its homologues. Firstly, the Lys45-Glu53 and Arg48-Glu53 interactions in scCK2α lead Lys50 to adopt a unique conformation that is able to stabilize the γ-phosphate of the co-substrate, which makes the existence of the `essential divalent cation' not so essential. The multiple nucleotide-divalent cation binding modes of the active site of scCK2α are apparently different from the two-divalent-cation-occupied active site of Zea mays CK2α and human CK2α. Secondly, conformational change of Glu53 in scCK2α-AMPPN breaks its interaction with Lys45 and Arg48; as a result, the co-substrate binding pocket becomes more open. This may suggest a clue to a possible ADP/GDP-release pathway, because the NE1 atom of the Trp in the `DWG motif' of CK2α forms a hydrogen bond to the O atom of Leu212, which seems to make ADP release by means of the `DFG-in flip to DFG-out' model found in most eukaryotic protein kinases impossible. Coincidentally, two sulfate ions which may mimic two phosphate groups were captured by Arg161 and Lys197 around the pocket. Mutagenesis and biochemical experiments on R161A and K197A mutants support the above proposal. Finally, scCK2α is unique in containing an insertion region whose function had not been identified in previous research. It is found that the insertion region contributes to maintaining the constitutively active conformation of the scCK2α catalytic site, but does not participate in interaction with the regulatory subunits.

VISA--Vector Integration Site Analysis server: a web-based server to rapidly identify retroviral integration sites from next-generation sequencing.

PubMed

Hocum, Jonah D; Battrell, Logan R; Maynard, Ryan; Adair, Jennifer E; Beard, Brian C; Rawlings, David J; Kiem, Hans-Peter; Miller, Daniel G; Trobridge, Grant D

2015-07-07

Analyzing the integration profile of retroviral vectors is a vital step in determining their potential genotoxic effects and developing safer vectors for therapeutic use. Identifying retroviral vector integration sites is also important for retroviral mutagenesis screens. We developed VISA, a vector integration site analysis server, to analyze next-generation sequencing data for retroviral vector integration sites. Sequence reads that contain a provirus are mapped to the human genome, sequence reads that cannot be localized to a unique location in the genome are filtered out, and then unique retroviral vector integration sites are determined based on the alignment scores of the remaining sequence reads. VISA offers a simple web interface to upload sequence files and results are returned in a concise tabular format to allow rapid analysis of retroviral vector integration sites.

A composite docking approach for the identification and characterization of ectosteric inhibitors of cathepsin K.

PubMed

Law, Simon; Panwar, Preety; Li, Jody; Aguda, Adeleke H; Jamroz, Andrew; Guido, Rafael V C; Brömme, Dieter

2017-01-01

Cathepsin K (CatK) is a cysteine protease that plays an important role in mammalian intra- and extracellular protein turnover and is known for its unique and potent collagenase activity. Through studies on the mechanism of its collagenase activity, selective ectosteric sites were identified that are remote from the active site. Inhibitors targeting these ectosteric sites are collagenase selective and do not interfere with other proteolytic activities of the enzyme. Potential ectosteric inhibitors were identified using a computational approach to screen the druggable subset of and the entire 281,987 compounds comprising Chemical Repository library of the National Cancer Institute-Developmental Therapeutics Program (NCI-DTP). Compounds were scored based on their affinity for the ectosteric site. Here we compared the scores of three individual molecular docking methods with that of a composite score of all three methods together. The composite docking method was up to five-fold more effective at identifying potent collagenase inhibitors (IC50 < 20 μM) than the individual methods. Of 160 top compounds tested in enzymatic assays, 28 compounds revealed blocking of the collagenase activity of CatK at 100 μM. Two compounds exhibited IC50 values below 5 μM corresponding to a molar protease:inhibitor concentration of <1:12. Both compounds were subsequently tested in osteoclast bone resorption assays where the most potent inhibitor, 10-[2-[bis(2-hydroxyethyl)amino]ethyl]-7,8-diethylbenzo[g]pteridine-2,4-dione, (NSC-374902), displayed an inhibition of bone resorption with an IC50-value of approximately 300 nM and no cell toxicity effects.

A composite docking approach for the identification and characterization of ectosteric inhibitors of cathepsin K

PubMed Central

Law, Simon; Panwar, Preety; Li, Jody; Aguda, Adeleke H.; Jamroz, Andrew; Guido, Rafael V. C.

2017-01-01

Cathepsin K (CatK) is a cysteine protease that plays an important role in mammalian intra- and extracellular protein turnover and is known for its unique and potent collagenase activity. Through studies on the mechanism of its collagenase activity, selective ectosteric sites were identified that are remote from the active site. Inhibitors targeting these ectosteric sites are collagenase selective and do not interfere with other proteolytic activities of the enzyme. Potential ectosteric inhibitors were identified using a computational approach to screen the druggable subset of and the entire 281,987 compounds comprising Chemical Repository library of the National Cancer Institute-Developmental Therapeutics Program (NCI-DTP). Compounds were scored based on their affinity for the ectosteric site. Here we compared the scores of three individual molecular docking methods with that of a composite score of all three methods together. The composite docking method was up to five-fold more effective at identifying potent collagenase inhibitors (IC50 < 20 μM) than the individual methods. Of 160 top compounds tested in enzymatic assays, 28 compounds revealed blocking of the collagenase activity of CatK at 100 μM. Two compounds exhibited IC50 values below 5 μM corresponding to a molar protease:inhibitor concentration of <1:12. Both compounds were subsequently tested in osteoclast bone resorption assays where the most potent inhibitor, 10-[2-[bis(2-hydroxyethyl)amino]ethyl]-7,8-diethylbenzo[g]pteridine-2,4-dione, (NSC-374902), displayed an inhibition of bone resorption with an IC50-value of approximately 300 nM and no cell toxicity effects. PMID:29088253

Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with Alpha-Alpha Domain Architecture that Catalyzes a Unique Cyclization-Fragmentation Reaction Sequence

PubMed Central

Harris, Golda G.; Lombardi, Patrick M.; Pemberton, Travis A.; Matsui, Tsutomu; Weiss, Thomas M.; Cole, Kathryn E.; Köksal, Mustafa; Murphy, Frank V.; Vedula, L. Sangeetha; Chou, Wayne K.W.; Cane, David E.; Christianson, David W.

2015-01-01

Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg2+ for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with 3 Mg2+ ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed based on ~36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis. PMID:26598179

Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1

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

T Nguyen; S Chang; I Evnouchidou

2011-12-31

ERAP1 trims antigen precursors to fit into MHC class I proteins. To fulfill this function, ERAP1 has unique substrate preferences, trimming long peptides but sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analyses suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of longmore » rather than short substrates induces a conformational change with reorientation of a key catalytic residue toward the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.« less

Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Stimulus-responsive zinc oxide-functionalized macromolecular humic acid nanocarrier for enhancement of antibacterial activity of ciprofloxacin hydrochloride.

PubMed

Murugesan, Gowri; Latha, Nachimuthu; Suganya, Kannan; Murugan, Marudhamuthu; Munusamy, Murugan A; Rajan, Mariappan

2018-07-15

Macromolecular of naturally occurring humic acid (HA) have garnered interest in the chemical, biological and medicine industry owing to their unique behavior, i.e., strong adsorptive and non-toxic nature. Here, we investigated the functionalization of organic (HA) with inorganic (ZnO) hybrid nanoparticles for topical and site-targeted delivery of ciprofloxacin by simple emulsification techniques. Ciprofloxacin (CIPRO)-encapsulated hybrid nanocarrier constitute an attractive novel drug delivery vehicle for sustained release of antibiotics to bacterial infection sites in an extended and controlled manner. The analytical characteristics of the designed system were thoroughly investigated by FTIR, XRD, SEM/EDAX, and TEM. The drug release of ciprofloxacin over 24h was 87.5%, 98.03%, 97.44%, and 97.24% for pH2.5, 5.5, 6.8, and 8.0, respectively. The antibacterial activities results confirmed that the CIPRO-encapsulated hybrid nanocarrier showed excellent growth inhibition against microorganisms. This hybrid nanocarrier loaded with antibiotics represents a promising approach for targeted and controlled drug delivery to infected sites. Copyright © 2018 Elsevier B.V. All rights reserved.

Optimizing Restriction Site Placement for Synthetic Genomes

NASA Astrophysics Data System (ADS)

Montes, Pablo; Memelli, Heraldo; Ward, Charles; Kim, Joondong; Mitchell, Joseph S. B.; Skiena, Steven

Restriction enzymes are the workhorses of molecular biology. We introduce a new problem that arises in the course of our project to design virus variants to serve as potential vaccines: we wish to modify virus-length genomes to introduce large numbers of unique restriction enzyme recognition sites while preserving wild-type function by substitution of synonymous codons. We show that the resulting problem is NP-Complete, give an exponential-time algorithm, and propose effective heuristics, which we show give excellent results for five sample viral genomes. Our resulting modified genomes have several times more unique restriction sites and reduce the maximum gap between adjacent sites by three to nine-fold.

Crystallographic comparison of manganese- and iron-dependent homoprotocatechuate 2,3-dioxygenases.

PubMed

Vetting, Matthew W; Wackett, Lawrence P; Que, Lawrence; Lipscomb, John D; Ohlendorf, Douglas H

2004-04-01

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four betaalphabetabetabeta modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 A), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An "open" coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.

Surface chemistry characterization of hydrodesulfurization and methanol synthesis model nanocatalysts

NASA Astrophysics Data System (ADS)

Komarneni, Mallikharjuna Rao

Surface science investigations of model catalysts have contributed significantly to heterogeneous catalysis over the past several decades. The unique properties of nanomaterials are being exploited in catalysis for the development of highly active and selective catalysts. Surface science investigations of model catalysts such as inorganic fullerene-like (IF) nanoparticles (NP), inorganic nanotubes (INT), and the oxide-supported nanoclusters are included in this dissertation. Thermal desorption spectroscopy and molecular beam scattering were respectively utilized to study the adsorption kinetics and dynamics of gas phase molecules on catalyst surfaces. In addition, ambient pressure kinetics experiments were performed to characterize the catalytic activity of hydrodesulfurization (HDS) nanocatalysts. The nanocatalysts were characterized with a variety of techniques, including Auger electron spectroscopy, x-ray photoelectron spectroscopy, electron microscopy, and x-ray diffraction. The adsorption kinetics studies of thiophene on novel HDS catalysts provided the first evidence for the presence of different adsorption sites on INT-WS2. Additionally, the adsorption sites on IF-MoS2 NP and silica-supported Mo clusters (Mo/silica) were characterized. Furthermore, the C-S bond activation energy of thiophene on Mo/silica was determined. These studies finally led to the fabrication of Ni/Co coated INT-WS2, which showed good catalytic activity towards HDS of thiophene. The studies of methanol synthesis catalysts include the adsorption kinetics and dynamics studies of CO and CO2 on Cu/silica and silica-supported EBL-fabricated Cu/CuOx nanoclusters. The adsorption dynamics of CO on Cu/silica are modeled within the frame work of the capture zone model (CZM), and the active sites of the silica-supported Au/Cu catalysts are successfully mapped. Studies on EBL model catalysts identify the rims of the CuOx nanoclusters as catalytically active sites. This observation has implications for new methanol catalyst design.

Crystal structure of KPC-2

PubMed Central

Ke, Wei; Bethel, Christopher R.; Thomson, Jodi M.; Bonomo, Robert A.; van den Akker, Focco

2008-01-01

β-lactamases inactivate β-lactam antibiotics and are a major cause of antibiotic resistance. The recent outbreaks of Klebsiella pneumoniae carbapenem-resistant (KPC) infections mediated by KPC type β-lactamases are creating a serious threat to our “last resort” antibiotics, the carbapenems. KPC β-lactamases are thus carbapenemases and are a subclass of Class A β-lactamases that have evolved to efficiently hydrolyze carbapenems and cephamycins which contain substitutions at the α position proximal to the carbonyl group that normally render these β-lactams resistant to hydrolysis. To investigate the molecular basis of this carbapenemase activity, we have determined the structure of KPC-2 at 1.85Å resolution. The active site of KPC-2 reveals the presence of a bicine buffer molecule which interacts via its carboxyl group with conserved active site residues S130, K234, T235, and T237; this likely resembles the interactions the β-lactam carboxyl moiety makes in the Michaelis-Menten complex. Comparison of the KPC-2 structure with non-carbapenemases and previously determined NMC-A and SME-1 carbapenemase structures shows several active site alterations that are unique among carbapenemases. An outward shift of the catalytic S70 residue renders the active sites of the carbapenemases more shallow likely allowing easier access of the bulkier substrates. Further space for the α-substituents is likely provided by shifts in N132 and N170 in addition to concerted movements in the postulated carboxyl binding pocket that might allow the substrates to bind in a slightly different angle to accommodate these α-substituents. The structure of KPC-2 thus provides key insights into the carbapenemase activity of emerging Class A β-lactamases. PMID:17441734

Stopover habitats of spring migrating surf scoters in southeast Alaska

USGS Publications Warehouse

Lok, E.K.; Esler, Daniel N.; Takekawa, John Y.; De La Cruz, S.W.; Sean, Boyd W.; Nysewander, D.R.; Evenson, J.R.; Ward, D.H.

2011-01-01

Habitat conditions and nutrient reserve levels during spring migration have been suggested as important factors affecting population declines in waterfowl, emphasizing the need to identify key sites used during spring and understand habitat features and resource availability at stopover sites. We used satellite telemetry to identify stopover sites used by surf scoters migrating through southeast Alaska during spring. We then contrasted habitat features of these sites to those of random sites to determine habitat attributes corresponding to use by migrating scoters. We identified 14 stopover sites based on use by satellite tagged surf scoters from several wintering sites. We identified Lynn Canal as a particularly important stopover site for surf scoters originating throughout the Pacific winter range; approximately half of tagged coastally migrating surf scoters used this site, many for extended periods. Stopover sites were farther from the mainland coast and closer to herring spawn sites than random sites, whereas physical shoreline habitat attributes were generally poor predictors of site use. The geography and resource availability within southeast Alaska provides unique and potentially critical stopover habitat for spring migrating surf scoters. Our work identifies specific sites and habitat resources that deserve conservation and management consideration. Aggregations of birds are vulnerable to human activity impacts such as contaminant spills and resource management decisions. This information is of value to agencies and organizations responsible for emergency response planning, herring fisheries management, and bird and ecosystem conservation.

Extending enzyme molecular recognition with an expanded amino acid alphabet

PubMed Central

Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam

2017-01-01

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894

Antiviral Activity of 3(2H)- and 6-Chloro-3(2H)-Isoflavenes against Highly Diverged, Neurovirulent Vaccine-Derived, Type2 Poliovirus Sewage Isolates

PubMed Central

Shulman, Lester M.; Sofer, Danit; Manor, Yossi; Mendelson, Ella; Balanant, Jean; Salvati, Anna Laura; Delpeyroux, Francis; Fiore, Lucia

2011-01-01

Background Substituted flavanoids interfere with uncoating of Enteroviruses including Sabin-2 polio vaccine strains. However flavanoid resistant and dependent, type-2 polio vaccine strains (minimally-diverged), emerged during in vitro infections. Between 1998–2009, highly-diverged (8 to >15%) type-2, aVDPV2s, from two unrelated persistent infections were periodically isolated from Israeli sewage. Aim To determine whether highly evolved aVDPV2s derived from persistent infections retained sensitivity to isoflavenes. Methods Sabin-2 and ten aVDPV2 isolates from two independent Israeli sources were titered on HEp2C cells in the presence and absence of 3(2H)- Isoflavene and 6-chloro-3(2H)-Isoflavene. Neurovirulence of nine aVDPV2s was measured in PVR-Tg-21 transgenic mice. Differences were related to unique amino acid substitutions within capsid proteins. Principal Findings The presence of either flavanoid inhibited viral titers of Sabin-2 and nine of ten aVDPV2s by one to two log10. The tenth aVDPV2, which had unique amino acid substitution distant from the isoflavene-binding pocket but clustered at the three- and five-fold axies of symmetry between capsomeres, was unaffected by both flavanoids. Genotypic neurovirulence attenuation sites in the 5′UTR and VP1 reverted in all aVDPV2s and all reacquired a full neurovirulent phenotype except one with amino acid substitutions flanking the VP1 site. Conclusion Both isoflavenes worked equally well against Sabin 2 and most of the highly-diverged, Israeli, aVDPV2s isolates. Thus, functionality of the hydrophobic pocket may be unaffected by selective pressures exerted during persistent poliovirus infections. Amino acid substitutions at sites remote from the drug-binding pocket and adjacent to a neurovirulence attenuation site may influence flavanoid antiviral activity, and neurovirulence, respectively. PMID:21904594

Nondestructive detection and measurement of hydrogen embrittlement

DOEpatents

Alex, Franklin; Byrne, Joseph Gerald

1977-01-01

A nondestructive system and method for the determination of the presence and extent of hydrogen embrittlement in metals, alloys, and other crystalline structures subject thereto. Positron annihilation characteristics of the positron-electron annihilation within the tested material provide unique energy distribution curves for each type of material tested at each respective stage of hydrogen embrittlement. Gamma radiation resulting from such annihilation events is detected and statistically summarized by appropriate instrumentation to reveal the variations of electron activity within the tested material caused by hydrogen embrittlement therein. Such data from controlled tests provides a direct indication of the relative stages of hydrogen embrittlement in the form of unique energy distribution curves which may be utilized as calibration curves for future comparison with field tests to give on-site indication of progressive stages of hydrogen embrittlement.

Computational evaluation of sub-nanometer cluster activity of singly exposed copper atom with various coordinative environment in catalytic CO2 transformation

NASA Astrophysics Data System (ADS)

Shanmugam, Ramasamy; Thamaraichelvan, Arunachalam; Ganesan, Tharumeya Kuppusamy; Viswanathan, Balasubramanian

2017-02-01

Metal cluster, at sub-nanometer level has a unique property in the activation of small molecules, in contrast to that of bulk surface. In the present work, singly exposed active site of copper metal cluster at sub-nanometer level was designed to arrive at the energy minimised configurations, binding energy, electrostatic potential map, frontier molecular orbitals and partial density of states. The ab initio molecular dynamics was carried out to probe the catalytic nature of the cluster. Further, the stability of the metal cluster and its catalytic activity in the electrochemical reduction of CO2 to CO were evaluated by means of computational hydrogen electrode via calculation of the free energy profile using DFT/B3LYP level of theory in vacuum. The activity of the cluster is ascertained from the fact that the copper atom, present in a two coordinative environment, performs a more selective conversion of CO2 to CO at an applied potential of -0.35 V which is comparatively lower than that of higher coordinative sites. The present study helps to design any sub-nano level metal catalyst for electrochemical reduction of CO2 to various value added chemicals.

Biodiversity on the Rocks: Macrofauna Inhabiting Authigenic Carbonate at Costa Rica Methane Seeps

PubMed Central

Levin, Lisa A.; Mendoza, Guillermo F.; Grupe, Benjamin M.; Gonzalez, Jennifer P.; Jellison, Brittany; Rouse, Greg; Thurber, Andrew R.; Waren, Anders

2015-01-01

Carbonate communities: The activity of anaerobic methane oxidizing microbes facilitates precipitation of vast quantities of authigenic carbonate at methane seeps. Here we demonstrate the significant role of carbonate rocks in promoting diversity by providing unique habitat and food resources for macrofaunal assemblages at seeps on the Costa Rica margin (400–1850 m). The attendant fauna is surprisingly similar to that in rocky intertidal shores, with numerous grazing gastropods (limpets and snails) as dominant taxa. However, the community feeds upon seep-associated microbes. Macrofaunal density, composition, and diversity on carbonates vary as a function of seepage activity, biogenic habitat and location. The macrofaunal community of carbonates at non-seeping (inactive) sites is strongly related to the hydrography (depth, temperature, O2) of overlying water, whereas the fauna at sites of active seepage is not. Densities are highest on active rocks from tubeworm bushes and mussel beds, particularly at the Mound 12 location (1000 m). Species diversity is higher on rocks exposed to active seepage, with multiple species of gastropods and polychaetes dominant, while crustaceans, cnidarians, and ophiuroids were better represented on rocks at inactive sites. Macro-infauna (larger than 0.3 mm) from tube cores taken in nearby seep sediments at comparable depths exhibited densities similar to those on carbonate rocks, but had lower diversity and different taxonomic composition. Seep sediments had higher densities of ampharetid, dorvilleid, hesionid, cirratulid and lacydoniid polychaetes, whereas carbonates had more gastropods, as well as syllid, chrysopetalid and polynoid polychaetes. Stable isotope signatures and metrics: The stable isotope signatures of carbonates were heterogeneous, as were the food sources and nutrition used by the animals. Carbonate δ13Cinorg values (mean = -26.98‰) ranged from -53.3‰ to +10.0‰, and were significantly heavier than carbonate δ13Corg (mean = -33.83‰), which ranged from -74.4‰ to -20.6‰. Invertebrates on carbonates had average δ13C (per rock) = -31.0‰ (range -18.5‰ to -46.5‰) and δ15N = 5.7‰ (range -4.5‰ to +13.4‰). Average δ13C values did not differ between active and inactive sites; carbonate fauna from both settings depend on chemosynthesis-based nutrition. Community metrics reflecting trophic diversity (SEAc, total Hull Area, ranges of δ13C and δ15N) and species packing (mean distance to centroid, nearest neighbor distance) also did not vary as a function of seepage activity or site. However, distinct isotopic signatures were observed among related, co-occurring species of gastropods and polychaetes, reflecting intense microbial resource partitioning. Overall, the substrate and nutritional heterogeneity introduced by authigenic seep carbonates act to promote diverse, uniquely adapted assemblages, even after seepage ceases. The macrofauna in these ecosystems remain largely overlooked in most surveys, but are major contributors to biodiversity of chemosynthetic ecosystems and the deep sea in general. PMID:26158723

Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response.

PubMed

Haoudi, Abdelali; Daniels, Rodney C; Wong, Eric; Kupfer, Gary; Semmes, O John

2003-09-26

The virally encoded oncoprotein Tax has been implicated in HTLV-1-mediated cellular transformation. The exact mechanism by which this protein contributes to the oncogenic process is not known. However, it has been hypothesized that Tax induces genomic instability via repression of cellular DNA repair. We examined the effect of de novo Tax expression upon the cell cycle, because appropriate activation of cell cycle checkpoints is essential to a robust damage-repair response. Upon induction of tax expression, Jurkat T-cells displayed a pronounced accumulation in G2/M that was reversible by caffeine. We examined the G2-specific checkpoint signaling response in these cells and found activation of the ATM/chk2-mediated pathway, whereas the ATR/chk1-mediated response was unaffected. Immunoprecipitation with anti-chk2 antibody results in co-precipitation of Tax demonstrating a direct interaction of Tax with a chk2-containing complex. We also show that Tax targets a discrete nuclear site and co-localizes with chk2 and not chk1. This nuclear site, previously identified as Tax Speckled Structures (TSS), also contains the early damage response factor 53BP1. The recruitment of 53BP1 to TSS is dependent upon ATM signaling and requires expression of Tax. Specifically, Tax expression induces redistribution of diffuse nuclear 53BP1 to the TSS foci. Taken together these data suggest that the TSS describe a unique nuclear site involved in DNA damage recognition, repair response, and cell cycle checkpoint activation. We suggest that association of Tax with this multifunctional subnuclear site results in disruption of a subset of the site-specific activities and contributes to cellular genomic instability.

Detailed glaciochemical investigations in southern Victoria Land - a proxy climatic record

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

Mayewski, P.A.

1987-09-01

Local accumulation-basins in the Transantarctic Mountains possess sites suitable for recovering ice-core records that are valuable for studying climate change. These sites are also unique, because they are close to the sites of other ice-core studies and to areas with established terrestrial records. The objective is to study a snowpit in detail and collect ice cores in southern Victoria Land; this work will be similar to the investigations that the authors has conducted in south Greenland and in the Dominion Range near the Beardmore Glacier. The proposed sites are in Convoy, Asgard, and Royal Society ranges. The authors will selectmore » one site at which he will recover two ice cores, each approximately 200 to 300 meters in depth. Samples will be analyzed for major anions (chloride, sulfate, nitrate, fluoride) and cations (sodium, potassium, magnesium, ammonium, silicate), total acidity, conductivity, density, and core stratigraphy with dating provided by cross-calibration of all of the preceding plus total beta-activity, lead-210, oxygen isotopes, and microparticles. This investigation will yield a detailed record of several thousand years of glacial history, climate change, and volcanic activity for southern Victoria Land. This record will be compared to existing terrestrial records to add necessary detail and to other global ice-core records to assess global climatic change. It will also help to document volcanic activity for Mount Erebus as well as other volcanos in the Southern Hemisphere and possibly some in the Northern Hemisphere. With this record, the author will be able to evaluate the influence of volcanic and solar activity on climate as well as add greatly to the understanding of the chemistry of the global atmosphere.« less

Bacterial Communities and the Nitrogen Cycle in the Gypsum Soils of Cuatro Ciénegas Basin, Coahuila: A Mars Analogue

PubMed Central

López-Lozano, Nguyen E.; Eguiarte, Luis E.; Bonilla-Rosso, Germán; García-Oliva, Felipe; Martínez-Piedragil, Celeste; Rooks, Christine

2012-01-01

Abstract The OMEGA/Mars Express hyperspectral imager identified gypsum at several sites on Mars in 2005. These minerals constitute a direct record of past aqueous activity and are important with regard to the search of extraterrestrial life. Gale Crater was chosen as Mars Science Laboratory Curiosity's landing site because it is rich in gypsum, as are some desert soils of the Cuatro Ciénegas Basin (CCB) (Chihuahuan Desert, Mexico). The gypsum of the CCB, which is overlain by minimal carbonate deposits, was the product of magmatic activity that occurred under the Tethys Sea. To examine this Mars analogue, we retrieved gypsum-rich soil samples from two contrasting sites with different humidity in the CCB. To characterize the site, we obtained nutrient data and analyzed the genes related to the N cycle (nifH, nirS, and nirK) and the bacterial community composition by using 16S rRNA clone libraries. As expected, the soil content for almost all measured forms of carbon, nitrogen, and phosphorus were higher at the more humid site than at the drier site. What was unexpected is the presence of a rich and divergent community at both sites, with higher taxonomic diversity at the humid site and almost no taxonomic overlap. Our results suggest that the gypsum-rich soils of the CCB host a unique microbial ecosystem that includes novel microbial assemblies. Key Words: Cuatro Ciénegas Basin—Gale Crater—Gypsum soil microbial diversity—Molecular ecology—Nitrogen cycle. Astrobiology 12, 699–709. PMID:22920518

Deepest and hottest hydrothermal activity in the Okinawa Trough: the Yokosuka site at Yaeyama Knoll

PubMed Central

Miyazaki, Junichi; Kawagucci, Shinsuke; Makabe, Akiko; Takahashi, Ayu; Kitada, Kazuya; Torimoto, Junji; Matsui, Yohei; Tasumi, Eiji; Shibuya, Takazo; Nakamura, Kentaro; Horai, Shunsuke; Sato, Shun; Ishibashi, Jun-ichiro; Kanzaki, Hayato; Nakagawa, Satoshi; Hirai, Miho; Takaki, Yoshihiro; Okino, Kyoko; Watanabe, Hiromi Kayama; Kumagai, Hidenori

2017-01-01

Since the initial discovery of hydrothermal vents in 1977, these ‘extreme’ chemosynthetic systems have been a focus of interdisciplinary research. The Okinawa Trough (OT), located in the semi-enclosed East China Sea between the Eurasian continent and the Ryukyu arc, hosts more than 20 known vent sites but all within a relatively narrow depth range (600–1880 m). Depth is a significant factor in determining fluid temperature and chemistry, as well as biological composition. However, due to the narrow depth range of known sites, the actual influence of depth here has been poorly resolved. Here, the Yokosuka site (2190 m), the first OT vent exceeding 2000 m depth is reported. A highly active hydrothermal vent site centred around four active vent chimneys reaching 364°C in temperature, it is the hottest in the OT. Notable Cl depletion (130 mM) and both high H2 and CH4 concentrations (approx. 10 mM) probably result from subcritical phase separation and thermal decomposition of sedimentary organic matter. Microbiota and fauna were generally similar to other sites in the OT, although with some different characteristics. In terms of microbiota, the H2-rich vent fluids in Neuschwanstein chimney resulted in the dominance of hydrogenotrophic chemolithoautotrophs such as Thioreductor and Desulfobacterium. For fauna, the dominance of the deep-sea mussel Bathymodiolus aduloides is surprising given other nearby vent sites are usually dominated by B. platifrons and/or B. japonicus, and a sponge field in the periphery dominated by Poecilosclerida is unusual for OT vents. Our insights from the Yokosuka site implies that although the distribution of animal species may be linked to depth, the constraint is perhaps not water pressure and resulting chemical properties of the vent fluid but instead physical properties of the surrounding seawater. The potential significance of these preliminary results and prospect for future research on this unique site are discussed. PMID:29308272

Deepest and hottest hydrothermal activity in the Okinawa Trough: the Yokosuka site at Yaeyama Knoll

NASA Astrophysics Data System (ADS)

Miyazaki, Junichi; Kawagucci, Shinsuke; Makabe, Akiko; Takahashi, Ayu; Kitada, Kazuya; Torimoto, Junji; Matsui, Yohei; Tasumi, Eiji; Shibuya, Takazo; Nakamura, Kentaro; Horai, Shunsuke; Sato, Shun; Ishibashi, Jun-ichiro; Kanzaki, Hayato; Nakagawa, Satoshi; Hirai, Miho; Takaki, Yoshihiro; Okino, Kyoko; Watanabe, Hiromi Kayama; Kumagai, Hidenori; Chen, Chong

2017-12-01

Since the initial discovery of hydrothermal vents in 1977, these `extreme' chemosynthetic systems have been a focus of interdisciplinary research. The Okinawa Trough (OT), located in the semi-enclosed East China Sea between the Eurasian continent and the Ryukyu arc, hosts more than 20 known vent sites but all within a relatively narrow depth range (600-1880 m). Depth is a significant factor in determining fluid temperature and chemistry, as well as biological composition. However, due to the narrow depth range of known sites, the actual influence of depth here has been poorly resolved. Here, the Yokosuka site (2190 m), the first OT vent exceeding 2000 m depth is reported. A highly active hydrothermal vent site centred around four active vent chimneys reaching 364°C in temperature, it is the hottest in the OT. Notable Cl depletion (130 mM) and both high H2 and CH4 concentrations (approx. 10 mM) probably result from subcritical phase separation and thermal decomposition of sedimentary organic matter. Microbiota and fauna were generally similar to other sites in the OT, although with some different characteristics. In terms of microbiota, the H2-rich vent fluids in Neuschwanstein chimney resulted in the dominance of hydrogenotrophic chemolithoautotrophs such as Thioreductor and Desulfobacterium. For fauna, the dominance of the deep-sea mussel Bathymodiolus aduloides is surprising given other nearby vent sites are usually dominated by B. platifrons and/or B. japonicus, and a sponge field in the periphery dominated by Poecilosclerida is unusual for OT vents. Our insights from the Yokosuka site implies that although the distribution of animal species may be linked to depth, the constraint is perhaps not water pressure and resulting chemical properties of the vent fluid but instead physical properties of the surrounding seawater. The potential significance of these preliminary results and prospect for future research on this unique site are discussed.

SusG: A Unique Cell-Membrane-Associated [alpha]-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

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

Koropatkin, Nicole M.; Smith, Thomas J.

SusG is an {alpha}-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysismore » demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.« less

Non-site-specific allosteric effect of oxygen on human hemoglobin under high oxygen partial pressure

PubMed Central

Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

2014-01-01

Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins. It is widely accepted that oxygen molecules (O2) act as a “site-specific” homotropic effector, or the successive O2 binding to the heme brings about the quaternary regulation. However, here we show that the site-specific allosteric effect is not necessarily only a unique mechanism of O2 allostery. Our simulation results revealed that the solution environment of high O2 partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional “non-site-specific” allosteric effect of O2. The latter effect should play a complementary role in the quaternary change by affecting the intersubunit contacts. This analysis must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view. PMID:24710521

Using the Textpresso Site-Specific Recombinases Web server to identify Cre expressing mouse strains and floxed alleles.

PubMed

Condie, Brian G; Urbanski, William M

2014-01-01

Effective tools for searching the biomedical literature are essential for identifying reagents or mouse strains as well as for effective experimental design and informed interpretation of experimental results. We have built the Textpresso Site Specific Recombinases (Textpresso SSR) Web server to enable researchers who use mice to perform in-depth searches of a rapidly growing and complex part of the mouse literature. Our Textpresso Web server provides an interface for searching the full text of most of the peer-reviewed publications that report the characterization or use of mouse strains that express Cre or Flp recombinase. The database also contains most of the publications that describe the characterization or analysis of strains carrying conditional alleles or transgenes that can be inactivated or activated by site-specific recombinases such as Cre or Flp. Textpresso SSR complements the existing online databases that catalog Cre and Flp expression patterns by providing a unique online interface for the in-depth text mining of the site specific recombinase literature.

Non-site-specific allosteric effect of oxygen on human hemoglobin under high oxygen partial pressure.

PubMed

Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

2014-04-08

Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins. It is widely accepted that oxygen molecules (O2) act as a "site-specific" homotropic effector, or the successive O2 binding to the heme brings about the quaternary regulation. However, here we show that the site-specific allosteric effect is not necessarily only a unique mechanism of O2 allostery. Our simulation results revealed that the solution environment of high O2 partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional "non-site-specific" allosteric effect of O2. The latter effect should play a complementary role in the quaternary change by affecting the intersubunit contacts. This analysis must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view.

Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

PubMed Central

Doolittle-Hall, Janet M.; Cunningham Glasspoole, Danielle L.; Seaman, William T.; Webster-Cyriaque, Jennifer

2015-01-01

Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures. PMID:26569308

Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes

PubMed Central

Li, Yongchao; Wang, Yang; Jiang, Haobo; Deng, Junpeng

2009-01-01

Arthropod phenoloxidase (PO) generates quinones and other toxic compounds to sequester and kill pathogens during innate immune responses. It is also involved in wound healing and other physiological processes. Insect PO is activated from its inactive precursor, prophenoloxidase (PPO), by specific proteolysis via a serine protease cascade. Here, we report the crystal structure of PPO from a lepidopteran insect at a resolution of 1.97 Å, which is the initial structure for a PPO from the type 3 copper protein family. Manduca sexta PPO is a heterodimer consisting of 2 homologous polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type 3 di-nuclear copper center, with each copper ion coordinated with 3 structurally conserved histidines. The acidic residue Glu-395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is key to the ortho-hydroxylase activity of PO. The structure provides unique insights into the mechanism by which type 3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced on cleavage at Arg-51 allows us to propose a model for localized PPO activation in insects. PMID:19805072

Ensuring a reliable satellite

NASA Astrophysics Data System (ADS)

Johnson, Charles E.; Persinger, Randy R.; Lemon, James J.; Volkert, Keith J.

Comprehensive testing and monitoring approaches have been formulated and implemented for Intelsat VI, which is the largest commercial satellite in service. An account is given of the ground test program from unit level through launch site activities, giving attention to the test data handling system. Test methods unique to Intelsat VI encompass near-field anechoic chamber antenna measurements, offloading 1-g deployment of solar cell and deflector antennas, and electrostatic discharge measurements. The problems accruing to the sheer size of this spacecraft are stressed.

Decision support methods for the environmental assessment of contamination at mining sites.

PubMed

Jordan, Gyozo; Abdaal, Ahmed

2013-09-01

Polluting mine accidents and widespread environmental contamination associated with historic mining in Europe and elsewhere has triggered the improvement of related environmental legislation and of the environmental assessment and management methods for the mining industry. Mining has some unique features such as natural background pollution associated with natural mineral deposits, industrial activities and contamination located in the three-dimensional sub-surface space, the problem of long-term remediation after mine closure, problem of secondary contaminated areas around mine sites and abandoned mines in historic regions like Europe. These mining-specific problems require special tools to address the complexity of the environmental problems of mining-related contamination. The objective of this paper is to review and evaluate some of the decision support methods that have been developed and applied to mining contamination. In this paper, only those methods that are both efficient decision support tools and provide a 'holistic' approach to the complex problem as well are considered. These tools are (1) landscape ecology, (2) industrial ecology, (3) landscape geochemistry, (4) geo-environmental models, (5) environmental impact assessment, (6) environmental risk assessment, (7) material flow analysis and (8) life cycle assessment. This unique inter-disciplinary study should enable both the researcher and the practitioner to obtain broad view on the state-of-the-art of decision support methods for the environmental assessment of contamination at mine sites. Documented examples and abundant references are also provided.

A cell wall-degrading esterase of Xanthomonas oryzae requires a unique substrate recognition module for pathogenesis on rice.

PubMed

Aparna, Gudlur; Chatterjee, Avradip; Sonti, Ramesh V; Sankaranarayanan, Rajan

2009-06-01

Xanthomonas oryzae pv oryzae (Xoo) causes bacterial blight, a serious disease of rice (Oryza sativa). LipA is a secretory virulence factor of Xoo, implicated in degradation of rice cell walls and the concomitant elicitation of innate immune responses, such as callose deposition and programmed cell death. Here, we present the high-resolution structural characterization of LipA that reveals an all-helical ligand binding module as a distinct functional attachment to the canonical hydrolase catalytic domain. We demonstrate that the enzyme binds to a glycoside ligand through a rigid pocket comprising distinct carbohydrate-specific and acyl chain recognition sites where the catalytic triad is situated 15 A from the anchored carbohydrate. Point mutations disrupting the carbohydrate anchor site or blocking the pocket, even at a considerable distance from the enzyme active site, can abrogate in planta LipA function, exemplified by loss of both virulence and the ability to elicit host defense responses. A high conservation of the module across genus Xanthomonas emphasizes the significance of this unique plant cell wall-degrading function for this important group of plant pathogenic bacteria. A comparison with the related structural families illustrates how a typical lipase is recruited to act on plant cell walls to promote virulence, thus providing a remarkable example of the emergence of novel functions around existing scaffolds for increased proficiency of pathogenesis during pathogen-plant coevolution.

The cost structure of routine infant immunization services: a systematic analysis of six countries

PubMed Central

Geng, Fangli; Suharlim, Christian; Brenzel, Logan; Resch, Stephen C; Menzies, Nicolas A

2017-01-01

Abstract Little information exists on the cost structure of routine infant immunization services in low- and middle-income settings. Using a unique dataset of routine infant immunization costs from six countries, we estimated how costs were distributed across budget categories and programmatic activities, and investigated how the cost structure of immunization sites varied by country and site characteristics. The EPIC study collected data on routine infant immunization costs from 319 sites in Benin, Ghana, Honduras, Moldova, Uganda, Zambia, using a standardized approach. For each country, we estimated the economic costs of infant immunization by administrative level, budget category, and programmatic activity from a programme perspective. We used regression models to describe how costs within each category were related to site operating characteristics and efficiency level. Site-level costs (incl. vaccines) represented 77–93% of national routine infant immunization costs. Labour and vaccine costs comprised 14–69% and 13–69% of site-level cost, respectively. The majority of site-level resources were devoted to service provision (facility-based or outreach), comprising 48–78% of site-level costs across the six countries. Based on the regression analyses, sites with the highest service volume had a greater proportion of costs devoted to vaccines, with vaccine costs per dose relatively unaffected by service volume but non-vaccine costs substantially lower with higher service volume. Across all countries, more efficient sites (compared with sites with similar characteristics) had a lower cost share devoted to labour. The cost structure of immunization services varied substantially between countries and across sites within each country, and was related to site characteristics. The substantial variation observed in this sample suggests differences in operating model for otherwise similar sites, and further understanding of these differences could reveal approaches to improve efficiency and performance of immunization sites. PMID:28575193

Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

PubMed Central

Koland, John G.

2014-01-01

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR (HER/ErbB) family receptors and growth factor receptor PTKs in general. PMID:24453959

Factors Associated With Time to Site Activation, Randomization, and Enrollment Performance in a Stroke Prevention Trial.

PubMed

Demaerschalk, Bart M; Brown, Robert D; Roubin, Gary S; Howard, Virginia J; Cesko, Eldina; Barrett, Kevin M; Longbottom, Mary E; Voeks, Jenifer H; Chaturvedi, Seemant; Brott, Thomas G; Lal, Brajesh K; Meschia, James F; Howard, George

2017-09-01

Multicenter clinical trials attempt to select sites that can move rapidly to randomization and enroll sufficient numbers of patients. However, there are few assessments of the success of site selection. In the CREST-2 (Carotid Revascularization and Medical Management for Asymptomatic Carotid Stenosis Trials), we assess factors associated with the time between site selection and authorization to randomize, the time between authorization to randomize and the first randomization, and the average number of randomizations per site per month. Potential factors included characteristics of the site, specialty of the principal investigator, and site type. For 147 sites, the median time between site selection to authorization to randomize was 9.9 months (interquartile range, 7.7, 12.4), and factors associated with early site activation were not identified. The median time between authorization to randomize and a randomization was 4.6 months (interquartile range, 2.6, 10.5). Sites with authorization to randomize in only the carotid endarterectomy study were slower to randomize, and other factors examined were not significantly associated with time-to-randomization. The recruitment rate was 0.26 (95% confidence interval, 0.23-0.28) patients per site per month. By univariate analysis, factors associated with faster recruitment were authorization to randomize in both trials, principal investigator specialties of interventional radiology and cardiology, pre-trial reported performance >50 carotid angioplasty and stenting procedures per year, status in the top half of recruitment in the CREST trial, and classification as a private health facility. Participation in StrokeNet was associated with slower recruitment as compared with the non-StrokeNet sites. Overall, selection of sites with high enrollment rates will likely require customization to align the sites selected to the factor under study in the trial. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02089217. © 2017 American Heart Association, Inc.

The cost structure of routine infant immunization services: a systematic analysis of six countries.

PubMed

Geng, Fangli; Suharlim, Christian; Brenzel, Logan; Resch, Stephen C; Menzies, Nicolas A

2017-10-01

Little information exists on the cost structure of routine infant immunization services in low- and middle-income settings. Using a unique dataset of routine infant immunization costs from six countries, we estimated how costs were distributed across budget categories and programmatic activities, and investigated how the cost structure of immunization sites varied by country and site characteristics. The EPIC study collected data on routine infant immunization costs from 319 sites in Benin, Ghana, Honduras, Moldova, Uganda, Zambia, using a standardized approach. For each country, we estimated the economic costs of infant immunization by administrative level, budget category, and programmatic activity from a programme perspective. We used regression models to describe how costs within each category were related to site operating characteristics and efficiency level. Site-level costs (incl. vaccines) represented 77-93% of national routine infant immunization costs. Labour and vaccine costs comprised 14-69% and 13-69% of site-level cost, respectively. The majority of site-level resources were devoted to service provision (facility-based or outreach), comprising 48-78% of site-level costs across the six countries. Based on the regression analyses, sites with the highest service volume had a greater proportion of costs devoted to vaccines, with vaccine costs per dose relatively unaffected by service volume but non-vaccine costs substantially lower with higher service volume. Across all countries, more efficient sites (compared with sites with similar characteristics) had a lower cost share devoted to labour. The cost structure of immunization services varied substantially between countries and across sites within each country, and was related to site characteristics. The substantial variation observed in this sample suggests differences in operating model for otherwise similar sites, and further understanding of these differences could reveal approaches to improve efficiency and performance of immunization sites. © The Author 2017. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine.

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

PubMed Central

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

2013-01-01

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

International Commission for Mountain Emergency Medicine Consensus Guidelines for On-Site Management and Transport of Patients in Canyoning Incidents.

PubMed

Strapazzon, Giacomo; Reisten, Oliver; Argenone, Fabien; Zafren, Ken; Zen-Ruffinen, Greg; Larsen, Gordon L; Soteras, Inigo

2018-06-01

Canyoning is a recreational activity that has increased in popularity in the last decade in Europe and North America, resulting in up to 40% of the total search and rescue costs in some geographic locations. The International Commission for Mountain Emergency Medicine convened an expert panel to develop recommendations for on-site management and transport of patients in canyoning incidents. The goal of the current review is to provide guidance to healthcare providers and canyoning rescue professionals about best practices for rescue and medical treatment through the evaluation of the existing best evidence, focusing on the unique combination of remoteness, water exposure, limited on-site patient management options, and technically challenging terrain. Recommendations are graded on the basis of quality of supporting evidence according to the classification scheme of the American College of Chest Physicians. Copyright © 2017 Wilderness Medical Society. Published by Elsevier Inc. All rights reserved.

Divergent expression of cytokinin biosynthesis, signaling and catabolism genes underlying differences in feeding sites induced by cyst and root-knot nematodes.

PubMed

Dowd, Carola D; Chronis, Demosthenis; Radakovic, Zoran S; Siddique, Shahid; Schmülling, Thomas; Werner, Tomáš; Kakimoto, Tatsuo; Grundler, Florian M W; Mitchum, Melissa G

2017-10-01

Cyst and root-knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root-knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Analysis of Substrate Access to Active Sites in Bacterial Multicomponent Monooxygenase Hydroxylases: X-ray Crystal Structure of Xenon-Pressurized Phenol Hydroxylase from Pseudomonas sp. OX1†,‡

PubMed Central

McCormick, Michael S.; Lippard, Stephen J.

2011-01-01

In all structurally characterized bacterial multicomponent monooxygenase (BMM) hydroxylase proteins, a series of hydrophobic cavities in the α-subunit trace a conserved path from the protein exterior to the carboxylate-bridged diiron active site. The present study examines these cavities as a potential route for dioxygen transport to the active site by crystallographic characterization of a xenon-pressurized sample of the hydroxylase component of phenol hydroxylase from Pseudomonas sp. OX1. Computational analyses of the hydrophobic cavities in the hydroxylase α-subunits of phenol hydroxylase (PHH), toluene/o-xylene monooxygenase (ToMOH), and soluble methane monooxygenase (sMMOH) are also presented. The results, together with previous findings from crystallographic studies of xenon-pressurized sMMO hydroxylase, clearly identify the propensity for these cavities to bind hydrophobic gas molecules in the protein interior. This proposed functional role is supported by recent stopped flow kinetic studies of ToMOH variants (Song, et al., 2011). In addition to information about the Xe sites, the structure determination revealed significantly reduced regulatory protein binding to the hydroxylase in comparison to the previously reported structure of PHH, as well as the presence of a newly identified metal binding site in the α-subunit that adopts a linear coordination environment consistent with Cu(I), and a glycerol molecule bound to Fe1 in a fashion that is unique among hydrocarbon-diiron site adducts reported to date in BMM hydroxylase structures. Finally, a comparative analysis of the α-subunit structures of MMOH, ToMOH, and PHH details proposed routes for the other three BMM substrates, the hydrocarbon, electrons, and protons, comprising cavities, channels, hydrogen-bonding networks, and pores in the structures of their α-subunits. PMID:22136180

Space Vehicle Valve System

NASA Technical Reports Server (NTRS)

Kelley, Anthony R. (Inventor); Lindner, Jeffrey L. (Inventor)

2014-01-01

The present invention is a space vehicle valve system which controls the internal pressure of a space vehicle and the flow rate of purged gases at a given internal pressure and aperture site. A plurality of quasi-unique variable dimension peaked valve structures cover the purge apertures on a space vehicle. Interchangeable sheet guards configured to cover valve apertures on the peaked valve structure contain a pressure-activated surface on the inner surface. Sheet guards move outwardly from the peaked valve structure when in structural contact with a purge gas stream flowing through the apertures on the space vehicle. Changing the properties of the sheet guards changes the response of the sheet guards at a given internal pressure, providing control of the flow rate at a given aperture site.

Epigenetic aspects of centromere function in plants.

PubMed

Birchler, James A; Gao, Zhi; Sharma, Anupma; Presting, Gernot G; Han, Fangpu

2011-04-01

Centromeres were once thought to be boring structures on the chromosome involved with transmission through mitosis and meiosis. Recent data from a wide spectrum of organisms reveal an epigenetic component to centromere specification in that they can become inactive easily or form over unique DNA as neocentromeres. However, the constancy of centromere repeats at primary constrictions in most species, the fact that these repeats are transcribed and incorporated into the kinetochore, and the phenomenon of reactivation of formerly inactive centromeres at the same chromosomal sites suggests some type of role of DNA sequence or configuration in establishing the site of kinetochores. Here we present evidence for epigenetic and structural aspects involved with centromere activity in plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals.

PubMed

Asakura, H; Makino, S; Kobori, H; Watarai, M; Shirahata, T; Ikeda, T; Takeshi, K

2001-08-01

Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans.

Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals.

PubMed Central

Asakura, H.; Makino, S.; Kobori, H.; Watarai, M.; Shirahata, T.; Ikeda, T.; Takeshi, K.

2001-01-01

Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans. PMID:11561972

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

PubMed

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

2016-07-15

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Reversible active site sulfoxygenation can explain the oxygen tolerance of a NAD+-reducing [NiFe] hydrogenase and its unusual infrared spectroscopic properties.

PubMed

Horch, Marius; Lauterbach, Lars; Mroginski, Maria Andrea; Hildebrandt, Peter; Lenz, Oliver; Zebger, Ingo

2015-02-25

Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H2 oxidation and production. The soluble NAD(+)-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H2 with the reduction of NAD(+) and displays a unique O2 tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained in vitro and in vivo could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O2 catalytically by means of an NADH-dependent (per)oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H2-conversion even in the presence of O2.

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

Huang, Pengxiang; Nedelcu, Daniel; Watanabe, Miyako

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition andmore » activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.« less

Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5*

PubMed Central

Liu, Liqiong; Richard, Jessica; Kim, Sunyoung; Wojcik, Edward J.

2014-01-01

Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable “next generation” target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are “druggable.” One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease. PMID:24737313

Assessing the challenges of multi-scope clinical research sites: an example from NIH HIV/AIDS clinical trials networks.

PubMed

Rosas, Scott R; Cope, Marie T; Villa, Christie; Motevalli, Mahnaz; Utech, Jill; Schouten, Jeffrey T

2014-04-01

Large-scale, multi-network clinical trials are seen as a means for efficient and effective utilization of resources with greater responsiveness to new discoveries. Formal structures instituted within the National Institutes of Health (NIH) HIV/AIDS Clinical Trials facilitate collaboration and coordination across networks and emphasize an integrated approach to HIV/AIDS vaccine, prevention and therapeutics clinical trials. This study examines the joint usage of clinical research sites as means of gaining efficiency, extending capacity, and adding scientific value to the networks. A semi-structured questionnaire covering eight clinical management domains was administered to 74 (62% of sites) clinical site coordinators at single- and multi-network sites to identify challenges and efficiencies related to clinical trials management activities and coordination with multi-network units. Overall, respondents at multi-network sites did not report more challenges than single-network sites, but did report unique challenges to overcome including in the areas of study prioritization, community engagement, staff education and training, and policies and procedures. The majority of multi-network sites reported that such affiliations do allow for the consolidation and cost-sharing of research functions. Suggestions for increasing the efficiency or performance of multi-network sites included streamlining standards and requirements, consolidating protocol activation methods, using a single cross-network coordinating centre, and creating common budget and payment mechanisms. The results of this assessment provide important information to consider in the design and management of multi-network configurations for the NIH HIV/AIDS Clinical Trials Networks, as well as others contemplating and promoting the concept of multi-network settings. © 2013 John Wiley & Sons Ltd.

Dynamic motif occupancy (DynaMO) analysis identifies transcription factors and their binding sites driving dynamic biological processes.

PubMed

Kuang, Zheng; Ji, Zhicheng; Boeke, Jef D; Ji, Hongkai

2018-01-09

Biological processes are usually associated with genome-wide remodeling of transcription driven by transcription factors (TFs). Identifying key TFs and their spatiotemporal binding patterns are indispensable to understanding how dynamic processes are programmed. However, most methods are designed to predict TF binding sites only. We present a computational method, dynamic motif occupancy analysis (DynaMO), to infer important TFs and their spatiotemporal binding activities in dynamic biological processes using chromatin profiling data from multiple biological conditions such as time-course histone modification ChIP-seq data. In the first step, DynaMO predicts TF binding sites with a random forests approach. Next and uniquely, DynaMO infers dynamic TF binding activities at predicted binding sites using their local chromatin profiles from multiple biological conditions. Another landmark of DynaMO is to identify key TFs in a dynamic process using a clustering and enrichment analysis of dynamic TF binding patterns. Application of DynaMO to the yeast ultradian cycle, mouse circadian clock and human neural differentiation exhibits its accuracy and versatility. We anticipate DynaMO will be generally useful for elucidating transcriptional programs in dynamic processes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of the catalytic activity of the EGF receptor

PubMed Central

Endres, Nicholas F.; Engel, Kate; Das, Rahul; Kovacs, Erika; Kuriyan, John

2011-01-01

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in cell growth that is often misregulated in cancer. Several recent studies highlight the unique structural mechanisms involved in its regulation. Some elucidate the important role that the juxtamembrane segment and the transmembrane helix play in stabilizing the activating asymmetric kinase dimer, and suggest that its activation mechanism is likely to be conserved amongst the other human EGFR-related receptors. Other studies provide new explanations for two long observed, but poorly understood phenomena, the apparent heterogeneity in ligand binding and the formation of ligand-independent dimers. New insights into the allosteric mechanisms utilized by intracellular regulators of EGFR provide hope that allosteric sites could be used as targets for drug development. PMID:21868214

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

PubMed

Narczyk, Marta; Bertoša, Branimir; Papa, Lucija; Vuković, Vedran; Leščić Ašler, Ivana; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija; Štefanić, Zoran

2018-04-01

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P i ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P i binding, while a three-site model is needed to characterize FA binding, irrespective of P i presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P i and FA shows, however, that P i binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P i moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463. © 2018 Federation of European Biochemical Societies.

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogues

PubMed Central

Lietzan, Adam D.; St. Maurice, Martin

2014-01-01

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates. PMID:24157795

Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures*

PubMed Central

Jain, Kanishk; Warmack, Rebeccah A.; Stavropoulos, Peter

2016-01-01

In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei. We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors. PMID:27387499

Sparse and Specific Coding during Information Transmission between Co-cultured Dentate Gyrus and CA3 Hippocampal Networks

PubMed Central

Poli, Daniele; Thiagarajan, Srikanth; DeMarse, Thomas B.; Wheeler, Bruce C.; Brewer, Gregory J.

2017-01-01

To better understand encoding and decoding of stimulus information in two specific hippocampal sub-regions, we isolated and co-cultured rat primary dentate gyrus (DG) and CA3 neurons within a two-chamber device with axonal connectivity via micro-tunnels. We tested the hypothesis that, in these engineered networks, decoding performance of stimulus site information would be more accurate when stimuli and information flow occur in anatomically correct feed-forward DG to CA3 vs. CA3 back to DG. In particular, we characterized the neural code of these sub-regions by measuring sparseness and uniqueness of the responses evoked by specific paired-pulse stimuli. We used the evoked responses in CA3 to decode the stimulation sites in DG (and vice-versa) by means of learning algorithms for classification (support vector machine, SVM). The device was placed over an 8 × 8 grid of extracellular electrodes (micro-electrode array, MEA) in order to provide a platform for monitoring development, self-organization, and improved access to stimulation and recording at multiple sites. The micro-tunnels were designed with dimensions 3 × 10 × 400 μm allowing axonal growth but not migration of cell bodies and long enough to exclude traversal by dendrites. Paired-pulse stimulation (inter-pulse interval 50 ms) was applied at 22 different sites and repeated 25 times in each chamber for each sub-region to evoke time-locked activity. DG-DG and CA3-CA3 networks were used as controls. Stimulation in DG drove signals through the axons in the tunnels to activate a relatively small set of specific electrodes in CA3 (sparse code). CA3-CA3 and DG-DG controls were less sparse in coding than CA3 in DG-CA3 networks. Using all target electrodes with the three highest spike rates (14%), the evoked responses in CA3 specified each stimulation site in DG with optimum uniqueness of 64%. Finally, by SVM learning, these evoked responses in CA3 correctly decoded the stimulation sites in DG for 43% of the trials, significantly higher than the reverse, i.e., how well-recording in DG could predict the stimulation site in CA3. In conclusion, our co-cultured model for the in vivo DG-CA3 hippocampal network showed sparse and specific responses in CA3, selectively evoked by each stimulation site in DG. PMID:28321182

Spatial Relation Between Left Atrial Anatomical Contact Areas and Circular Activation in Persistent Atrial Fibrillation.

PubMed

Nakahara, Shiro; Yamaguchi, Takanori; Hori, Yuichi; Anjo, Naofumi; Hayashi, Akiko; Kobayashi, Sayuki; Komatsu, Takaaki; Sakai, Yoshihiko; Fukui, Akira; Tsuchiya, Takeshi; Taguchi, Isao

2016-05-01

Atrial low-voltage zones (LVZs) may be related to maintenance of atrial fibrillation (AF). The influence of left atrial (LA) contact areas (CoAs) on reentrant or rotor-like sources maintaining AF has not been investigated. Forty patients with persistent AF (PsAF) were analyzed. Three representative CoA regions in the LA (ascending aorta: anterior wall; descending aorta: left inferior pulmonary vein; and vertebrae: posterior wall) were visualized by enhanced CT. Using circular catheters, the LVZs (<0.5 mV) were assessed after restoration of SR, and local activation mapping and frequency domain analyses were performed after induction of AF. Circular activation during AF was visually defined as sites with ≥2 rotations by serial electrograms encompassing >80% of the mean AF cycle length. A pivot was defined as the core of the localized circular activation. Anterior (39/40 patients, 98%), left pulmonary vein antrum (27/40, 68%), and posterior (19/40, 48%) CoAs were identified, and 80% (68/85) of those sites were overlapped by or close (<3 mm) to LVZs. Thirty-six (90%) patients demonstrated circular activation (3.1±1.7 sites/patients) along with significantly higher organized dominant frequencies (6.3 ± 0.5 Hz, regularity-index: 0.26 [0.23-0.41]) within the LA, and the average electrogram amplitude of those pivots was 0.30 mV (0.18-0.52). Of those sites, 55% (66/120) were located at or close to CoA regions. Catheter ablation including of LVZs neighboring CoAs terminated AF in 9 (23%) patients. External anatomical structures contacting the LA may be related to unique conduction properties in diseased myocardium necessary for PsAF maintenance. © 2016 Wiley Periodicals, Inc.

Confederated Tribes Umatilla Indian Reservation (CTUIR) Umatilla Anadromous Fisheries Habitat Project : A Columbia River Basin Fish Habitat Project : Annual Report Fiscal Year 2007.

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

Hoverson, Eric D.; Amonette, Alexandra

2008-12-02

The Umatilla Anadromous Fisheries Habitat Project (UAFHP) is an ongoing effort to protect, enhance, and restore riparian and instream habitat for the natural production of anadromous salmonids in the Umatilla River Basin, Northeast Oregon. Flow quantity, water temperature, passage, and lack of in-stream channel complexity have been identified as the key limiting factors in the basin. During the 2007 Fiscal Year (FY) reporting period (February 1, 2007-January 31, 2008) primary project activities focused on improving instream and riparian habitat complexity, migrational passage, and restoring natural channel morphology and floodplain function. Eight fisheries habitat enhancement projects were implemented on Meacham Creek,more » Camp Creek, Greasewood Creek, Birch Creek, West Birch Creek, and the Umatilla River. Specific restoration actions included: (1) rectifying five fish passage barriers on four creeks, (2) planting 1,275 saplings and seeding 130 pounds of native grasses, (3) constructing two miles of riparian fencing for livestock exclusion, (4) coordinating activities related to the installation of two off-channel, solar-powered watering areas for livestock, and (5) developing eight water gap access sites to reduce impacts from livestock. Baseline and ongoing monitoring and evaluation activities were also completed on major project areas such as conducting photo point monitoring strategies activities at the Meacham Creek Large Wood Implementation Project site (FY2006) and at all existing easements and planned project sites. Fish surveys and aquatic habitat inventories were conducted at project sites prior to implementation. Monitoring plans will continue throughout the life of each project to oversee progression and inspire timely managerial actions. Twenty-seven conservation easements were maintained with 23 landowners. Permitting applications for planned project activities and biological opinions were written and approved. Project activities were based on a variety of fisheries monitoring techniques and habitat assessments used to determine existing conditions and identify factors limiting anadromous salmonid abundance. Proper selection and implementation of the most effective site-specific habitat restoration plan, taking into consideration the unique characteristics of each project site, and conducted in cooperation with landowners and project partners, was of paramount importance to ensure each project's success.« less

Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase.

PubMed

Fecker, Tobias; Galaz-Davison, Pablo; Engelberger, Felipe; Narui, Yoshie; Sotomayor, Marcos; Parra, Loreto P; Ramírez-Sarmiento, César A

2018-03-27

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA)

PubMed Central

2011-01-01

Background Staphylococcus aureus is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity. Results NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates. Conclusion Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing S. aureus with the ability to modulate host immune responses. PMID:22035583

Slow-Theta-to-Gamma Phase–Amplitude Coupling in Human Hippocampus Supports the Formation of New Episodic Memories

PubMed Central

Lega, Bradley; Burke, John; Jacobs, Joshua; Kahana, Michael J.

2016-01-01

Phase–amplitude coupling (PAC) has been proposed as a neural mechanism for coordinating information processing across brain regions. Here we sought to characterize PAC in the human hippocampus, and in temporal and frontal cortices, during the formation of new episodic memories. Intracranial recordings taken as 56 neurosurgical patients studied and recalled lists of words revealed significant hippocampal PAC, with slow-theta activity (2.5–5 Hz) modulating gamma band activity (34–130 Hz). Furthermore, a significant number of hippocampal electrodes exhibited greater PAC during successful than unsuccessful encoding, with the gamma activity at these sites coupled to the trough of the slow-theta oscillation. These same conditions facilitate LTP in animal models, providing a possible mechanism of action for this effect in human memory. Uniquely in the hippocampus, phase preference during item encoding exhibited a biphasic pattern. Overall, our findings help translate between the patterns identified during basic memory tasks in animals and those present during complex human memory encoding. We discuss the unique properties of human hippocampal PAC and how our findings relate to influential theories of information processing based on theta–gamma interactions. PMID:25316340

Simultaneous structure-activity studies and arming of natural products by C-H amination reveal cellular targets of eupalmerin acetate.

PubMed

Li, Jing; Cisar, Justin S; Zhou, Cong-Ying; Vera, Brunilda; Williams, Howard; Rodríguez, Abimael D; Cravatt, Benjamin F; Romo, Daniel

2013-06-01

Natural products have a venerable history of, and enduring potential for the discovery of useful biological activity. To fully exploit this, the development of chemical methodology that can functionalize unique sites within these complex structures is highly desirable. Here, we describe the use of rhodium(II)-catalysed C-H amination reactions developed by Du Bois to carry out simultaneous structure-activity relationship studies and arming (alkynylation) of natural products at 'unfunctionalized' positions. Allylic and benzylic C-H bonds in the natural products undergo amination while olefins undergo aziridination, and tertiary amine-containing natural products are converted to amidines by a C-H amination-oxidation sequence or to hydrazine sulfamate zwitterions by an unusual N-amination. The alkynylated derivatives are ready for conversion into cellular probes that can be used for mechanism-of-action studies. Chemo- and site-selectivity was studied with a diverse library of natural products. For one of these-the marine-derived anticancer diterpene, eupalmerin acetate-quantitative proteome profiling led to the identification of several protein targets in HL-60 cells, suggesting a polypharmacological mode of action.

Simultaneous structure-activity studies and arming of natural products by C-H amination reveal cellular targets of eupalmerin acetate

NASA Astrophysics Data System (ADS)

Li, Jing; Cisar, Justin S.; Zhou, Cong-Ying; Vera, Brunilda; Williams, Howard; Rodríguez, Abimael D.; Cravatt, Benjamin F.; Romo, Daniel

2013-06-01

Natural products have a venerable history of, and enduring potential for the discovery of useful biological activity. To fully exploit this, the development of chemical methodology that can functionalize unique sites within these complex structures is highly desirable. Here, we describe the use of rhodium(II)-catalysed C-H amination reactions developed by Du Bois to carry out simultaneous structure-activity relationship studies and arming (alkynylation) of natural products at ‘unfunctionalized’ positions. Allylic and benzylic C-H bonds in the natural products undergo amination while olefins undergo aziridination, and tertiary amine-containing natural products are converted to amidines by a C-H amination-oxidation sequence or to hydrazine sulfamate zwitterions by an unusual N-amination. The alkynylated derivatives are ready for conversion into cellular probes that can be used for mechanism-of-action studies. Chemo- and site-selectivity was studied with a diverse library of natural products. For one of these—the marine-derived anticancer diterpene, eupalmerin acetate—quantitative proteome profiling led to the identification of several protein targets in HL-60 cells, suggesting a polypharmacological mode of action.

Structural and mechanistic insights into Mps1 kinase activation

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

Wang, Wei; Yang, Yuting; Gao, Yuefeng

2010-11-05

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-{angstrom}-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the {alpha}C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation.more » Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices {alpha}EF and {alpha}F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.« less

The STAT3-Ser/Hes3 signaling axis: an emerging regulator of endogenous regeneration and cancer growth

PubMed Central

Poser, Steven W.; Park, Deric M.; Androutsellis-Theotokis, Andreas

2013-01-01

Stem cells, by definition, are able to both self-renew (give rise to more cells of their own kind) and demonstrate multipotential (the ability to differentiate into multiple cell types). To accommodate this unique dual ability, stem cells interpret signal transduction pathways in specialized ways. Notable examples include canonical and non-canonical branches of the Notch signaling pathway, with each controlling different downstream targets (e.g., Hes1 vs. Hes3) and promoting either differentiation or self-renewal. Similarly, stem cells utilize STAT3 signaling uniquely. Most mature cells studied thus far rely on tyrosine phosphorylation (STAT3-Tyr) to promote survival and growth; in contrast, STAT3-Tyr induces the differentiation of neural stem cells (NSCs). NSCs use an alternative phosphorylation site, STAT3-Ser, to regulate survival and growth, a site that is largely redundant for this function in most other cell types. STAT3-Ser regulates Hes3, and together they form a convergence point for several signals, including Notch, Tie2, and insulin receptor activation. Disregulation and manipulation of the STAT3-Ser/Hes3 signaling pathway is important in both tumorigenesis and regenerative medicine, and worthy of extensive study. PMID:24101906

Framework for Site Characterization for Monitored Natural Attenuation of Volatile Organic Compounds in Ground Water

EPA Science Inventory

Monitored Natural Attenuation (MNA) is unique among remedial technologies in relying entirely on natural processes to achieve site-specific objectives. Site characterization is essential to provide site-specific data and interpretations for the decision-making process (i.e., to ...

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

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

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites onmore » the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.« less

Effect of 10.5 M Aqueous Urea on Helicobacter pylori Urease: A Molecular Dynamics Study.

PubMed

Minkara, Mona S; Weaver, Michael N; Merz, Kenneth M

2015-07-07

The effects of a 10.5 M solution of aqueous urea on Helicobacter pylori urease were investigated over the course of a 500 ns molecular dynamics (MD) simulation. The enzyme was solvated by 25321 water molecules, and additionally, 4788 urea molecules were added to the solution. Although concentrated urea solutions are known laboratory denaturants, the protein secondary structure is retained throughout the simulation largely because of the short simulation time (urea denaturation occurs on the millisecond time scale). The relatively constant solvent accessible surface area over the last 400 ns of the simulation further confirms the overall lack of denaturation. The wide-open flap state observed previously in Klebsiella areogenes urease [Roberts, B. P., et al. (2012) J. Am. Chem. Soc. 134, 9934] and H. pylori [Minkara, M. S., et al. (2014) J. Chem. Theory Comput. 10, 1852-1862] was also identified in this aqueous urea simulation. Over the course of the trajectory, we were able to observe urea molecules entering the active site in proportions related to the extent of opening of the active site-covering flap. Furthermore, urea molecules were observed to approach the pentacoordinate Ni(2+) ion in position to bind in a manner consistent with the proposed initial coordination step of the hydrolysis mechanism. We also observed a specific and unique pattern in the regions of the protein with a high root-mean-square fluctuation (rmsf). The high-rmsf regions in the β-chain form a horseshoelike arrangement surrounding the active site-covering flap on the surface of the protein. We hypothesize that the function of these regions is to both attract and shuttle urea toward the loop of the active site-covering flap before entry into the cavity. Indeed, urea is observed to interact with these regions for extended periods of simulation time before active site ingress.

Specificity determinants for autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis.

PubMed

Mo, Charlie Y; Birdwell, L Dillon; Kohli, Rahul M

2014-05-20

Bacteria utilize the tightly regulated stress response (SOS) pathway to respond to a variety of genotoxic agents, including antimicrobials. Activation of the SOS response is regulated by a key repressor-protease, LexA, which undergoes autoproteolysis in the setting of stress, resulting in derepression of SOS genes. Remarkably, genetic inactivation of LexA's self-cleavage activity significantly decreases acquired antibiotic resistance in infection models and renders bacteria hypersensitive to traditional antibiotics, suggesting that a mechanistic study of LexA could help inform its viability as a novel target for combating acquired drug resistance. Despite structural insights into LexA, a detailed knowledge of the enzyme's protease specificity is lacking. Here, we employ saturation and positional scanning mutagenesis on LexA's internal cleavage region to analyze >140 mutants and generate a comprehensive specificity profile of LexA from the human pathogen Pseudomonas aeruginosa (LexAPa). We find that the LexAPa active site possesses a unique mode of substrate recognition. Positions P1-P3 prefer small hydrophobic residues that suggest specific contacts with the active site, while positions P5 and P1' show a preference for flexible glycine residues that may facilitate the conformational change that permits autoproteolysis. We further show that stabilizing the β-turn within the cleavage region enhances LexA autoproteolytic activity. Finally, we identify permissive positions flanking the scissile bond (P4 and P2') that are tolerant to extensive mutagenesis. Our studies shed light on the active site architecture of the LexA autoprotease and provide insights that may inform the design of probes of the SOS pathway.

SUMMARY OF TECHNIQUES AND UNIQUE USES FOR DIRECT PUSH METHODS IN SITE CHARACTERIZATION ON CONTAMINATED FIELD SITES

EPA Science Inventory

Site characterization of subsurface contaminant transport is often hampered by a lack of knowledge of site heterogeneity and temporal variations in hydrogeochemistry. Two case studies are reviewed to illustrate the utility of macro-scale mapping information along with spatially-...

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase

PubMed Central

Heyes, Derren J.; Levy, Colin; Lafite, Pierre; Roberts, Ian S.; Goldrick, Marie; Stachulski, Andrew V.; Rossington, Steven B.; Stanford, Deborah; Rigby, Stephen E. J.; Scrutton, Nigel S.; Leys, David

2009-01-01

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2β-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2β-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg2+ ion (Mg-B). Both ligands occupy conformations compatible with an Sn2-type attack on the α-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg2+ ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp100 and Asp235 in addition to the CTP α-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn2+-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg2+ to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the α-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group. PMID:19815542

Ub-ISAP: a streamlined UNIX pipeline for mining unique viral vector integration sites from next generation sequencing data.

PubMed

Kamboj, Atul; Hallwirth, Claus V; Alexander, Ian E; McCowage, Geoffrey B; Kramer, Belinda

2017-06-17

The analysis of viral vector genomic integration sites is an important component in assessing the safety and efficiency of patient treatment using gene therapy. Alongside this clinical application, integration site identification is a key step in the genetic mapping of viral elements in mutagenesis screens that aim to elucidate gene function. We have developed a UNIX-based vector integration site analysis pipeline (Ub-ISAP) that utilises a UNIX-based workflow for automated integration site identification and annotation of both single and paired-end sequencing reads. Reads that contain viral sequences of interest are selected and aligned to the host genome, and unique integration sites are then classified as transcription start site-proximal, intragenic or intergenic. Ub-ISAP provides a reliable and efficient pipeline to generate large datasets for assessing the safety and efficiency of integrating vectors in clinical settings, with broader applications in cancer research. Ub-ISAP is available as an open source software package at https://sourceforge.net/projects/ub-isap/ .

Non-homeodomain regions of Hox proteins mediate activation versus repression of Six2 via a single enhancer site in vivo

PubMed Central

Yallowitz, Alisha R.; Gong, Ke-Qin; Swinehart, Ilea T.; Nelson, Lisa T.; Wellik, Deneen M.

2009-01-01

Summary Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity is poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins. PMID:19716816

HIV integration sites in latently infected cell lines: evidence of ongoing replication.

PubMed

Symons, Jori; Chopra, Abha; Malatinkova, Eva; De Spiegelaere, Ward; Leary, Shay; Cooper, Don; Abana, Chike O; Rhodes, Ajantha; Rezaei, Simin D; Vandekerckhove, Linos; Mallal, Simon; Lewin, Sharon R; Cameron, Paul U

2017-01-13

Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

Electrochemical sensors and biosensors based on less aggregated graphene.

PubMed

Bo, Xiangjie; Zhou, Ming; Guo, Liping

2017-03-15

As a novel single-atom-thick sheet of sp 2 hybridized carbon atoms, graphene (GR) has attracted extensive attention in recent years because of its unique and remarkable properties, such as excellent electrical conductivity, large theoretical specific surface area, and strong mechanical strength. However, due to the π-π interaction, GR sheets are inclined to stack together, which may seriously degrade the performance of GR with the unique single-atom layer. In recent years, an increasing number of GR-based electrochemical sensors and biosensors are reported, which may reflect that GR has been considered as a kind of hot and promising electrode material for electrochemical sensor and biosensor construction. However, the active sites on GR surface induced by the irreversible GR aggregations would be deeply secluded inside the stacked GR sheets and therefore are not available for the electrocatalysis. So the alleviation or the minimization of the aggregation level for GR sheets would facilitate the exposure of active sites on GR and effectively upgrade the performance of GR-based electrochemical sensors and biosensors. Less aggregated GR with low aggregation and high dispersed structure can be used in improving the electrochemical activity of GR-based electrochemical sensors or biosensors. In this review, we summarize recent advances and new progress for the development of electrochemical sensors based on less aggregated GR. To achieve such goal, many strategies (such as the intercalation of carbon materials, surface modification, and structural engineering) have been applied to alleviate the aggregation level of GR in order to enhance the performance of GR-based electrochemical sensors and biosensors. Finally, the challenges associated with less aggregated GR-based electrochemical sensors and biosensors as well as related future research directions are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Common occupational classification system - revision 3

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

Stahlman, E.J.; Lewis, R.E.

1996-05-01

Workforce planning has become an increasing concern within the DOE community as the Office of Environmental Restoration and Waste Management (ER/WM or EM) seeks to consolidate and refocus its activities and the Office of Defense Programs (DP) closes production sites. Attempts to manage the growth and skills mix of the EM workforce while retaining the critical skills of the DP workforce have been difficult due to the lack of a consistent set of occupational titles and definitions across the complex. Two reasons for this difficulty may be cited. First, classification systems commonly used in industry often fail to cover inmore » sufficient depth the unique demands of DOE`s nuclear energy and research community. Second, the government practice of contracting the operation of government facilities to the private sector has introduced numerous contractor-specific classification schemes to the DOE complex. As a result, sites/contractors report their workforce needs using unique classification systems. It becomes difficult, therefore, to roll these data up to the national level necessary to support strategic planning and analysis. The Common Occupational Classification System (COCS) is designed to overcome these workforce planning barriers. The COCS is based on earlier workforce planning activities and the input of technical, workforce planning, and human resource managers from across the DOE complex. It provides a set of mutually-exclusive occupation titles and definitions that cover the broad range of activities present in the DOE complex. The COCS is not a required record-keeping or data management guide. Neither is it intended to replace contractor/DOE-specific classification systems. Instead, the system provides a consistent, high- level, functional structure of occupations to which contractors can crosswalk (map) their job titles.« less

Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

PubMed Central

Friedman, Joshua I.; Stivers, James T.

2010-01-01

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

Surface chemistry and catalytic performance of amorphous NiB/Hβ catalyst for n-hexane isomerization

NASA Astrophysics Data System (ADS)

Chen, Jinshe; Cai, Tingting; Jing, Xiaohui; Zhu, Lijun; Zhou, Yulu; Xiang, Yuzhi; Xia, Daohong

2016-12-01

The amorphous NiB nanoparticles were synthesized and a novel type of NiB/Hβ catalyst was prepared for the isomerization of n-hexane. The optimum preparation conditions were investigated and the effect of preparation conditions on the surface chemistry information of catalysts was characterized by XRD, N2 sorption studies, XPS, TPD and other related means. It was demonstrated that the loading amounts of NiB have effect on textural properties and the acid properties of surface. The loading amounts of NiB were also related to the amount of strong Lewis acid sites and the ratios of weak acid to strong acid of samples. Meanwhile, calcination temperatures of samples were closely associated with the structure of active components that function as metal centers. When the loading amount of NiB was 5 wt.% and calcination temperature was 200 °C, the catalyst had proper surface acidity sites and metal active sites to provide suitable synergistic effects. The mechanism for n-hexane isomerization was also investigated and the existence of unique structure of Bsbnd Nisbnd H was proved, which could provide good hydrogenation-dehydrogenation functions.

Biological Evaluation and X-ray Co-crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase, an Enzyme from the Redox Metabolism of Trypanosoma.

PubMed

De Gasparo, Raoul; Brodbeck-Persch, Elke; Bryson, Steve; Hentzen, Nina B; Kaiser, Marcel; Pai, Emil F; Krauth-Siegel, R Luise; Diederich, François

2018-05-08

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (K i ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC 50 ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

PubMed Central

Yeung, A T; Mattes, W B; Grossman, L

1986-01-01

An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction. PMID:3960727

2D Metal Chalcogenides Incorporated into Carbon and their Assembly for Energy Storage Applications.

PubMed

Deng, Zongnan; Jiang, Hao; Li, Chunzhong

2018-05-01

2D metal chalcogenides have become a popular focus in the energy storage field because of their unique properties caused by their single-atom thicknesses. However, their high surface energy and van der Waals attraction easily cause serious stacking and restacking, leading to the generation of more inaccessible active sites with rapid capacity fading. The hybridization of 2D metal chalcogenides with highly conductive materials, particularly, incorporating ultrasmall and few-layered metal chalcogenides into carbon frameworks, can not only maximize the exposure of active sites but also effectively avoid their stacking and aggregation during the electrochemical reaction process. Therefore, a satisfactory specific capacity will be achieved with a long cycle life. In this Concept, the representative progress on such intriguing nanohybrids and their applications in energy storage devices are mainly summarized. Finally, an outlook of the future development and challenges of such nanohybrids for achieving an excellent energy storage capability is also provided. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell fate regulation governed by a repurposed bacterial histidine kinase

DOE PAGES

Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; ...

2014-10-28

One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interactionmore » between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.« less

Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

PubMed

Ryan, Daniel E; Taussig, David; Steinfeld, Israel; Phadnis, Smruti M; Lunstad, Benjamin D; Singh, Madhurima; Vuong, Xuan; Okochi, Kenji D; McCaffrey, Ryan; Olesiak, Magdalena; Roy, Subhadeep; Yung, Chong Wing; Curry, Bo; Sampson, Jeffrey R; Bruhn, Laurakay; Dellinger, Douglas J

2018-01-25

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs

PubMed Central

Ryan, Daniel E; Taussig, David; Steinfeld, Israel; Phadnis, Smruti M; Lunstad, Benjamin D; Singh, Madhurima; Vuong, Xuan; Okochi, Kenji D; McCaffrey, Ryan; Olesiak, Magdalena; Roy, Subhadeep; Yung, Chong Wing; Curry, Bo; Sampson, Jeffrey R; Dellinger, Douglas J

2018-01-01

Abstract CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence (‘guide sequence’) and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications. PMID:29216382

Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Wurtz, Jean Marie; Bonnefond, Luc; Cavarelli, Jean

2014-09-01

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

Dual-stimuli responsive and reversibly activatable theranostic nanoprobe for precision tumor-targeting and fluorescence-guided photothermal therapy

NASA Astrophysics Data System (ADS)

Zhao, Xu; Yang, Cheng-Xiong; Chen, Li-Gong; Yan, Xiu-Ping

2017-05-01

The integrated functions of diagnostics and therapeutics make theranostics great potential for personalized medicine. Stimulus-responsive therapy allows spatial control of therapeutic effect only in the site of interest, and offers promising opportunities for imaging-guided precision therapy. However, the imaging strategies in previous stimulus-responsive therapies are `always on' or irreversible `turn on' modality, resulting in poor signal-to-noise ratios or even `false positive' results. Here we show the design of dual-stimuli-responsive and reversibly activatable nanoprobe for precision tumour-targeting and fluorescence-guided photothermal therapy. We fabricate the nanoprobe from asymmetric cyanine and glycosyl-functionalized gold nanorods (AuNRs) with matrix metalloproteinases (MMPs)-specific peptide as a linker to achieve MMPs/pH synergistic and pH reversible activation. The unique activation and glycosyl targetibility makes the nanoprobe bright only in tumour sites with negligible background, while AuNRs and asymmetric cyanine give synergistic photothermal effect. This work paves the way to designing efficient nanoprobes for precision theranostics.

Factors affecting female space use in ten populations of prairie chickens

USGS Publications Warehouse

Winder, Virginia L.; Carrlson, Kaylan M.; Gregory, Andrew J.; Hagen, Christian A.; Haukos, David A.; Kesler, Dylan C.; Larsson, Lena C.; Matthews, Ty W.; McNew, Lance B.; Patten, Michael; Pitman, Jim C.; Powell, Larkin A.; Smith, Jennifer A.; Thompson, Tom; Wolfe, Donald H.; Sandercock, Brett K.

2015-01-01

Conservation of wildlife depends on an understanding of the interactions between animal movements and key landscape factors. Habitat requirements of wide-ranging species often vary spatially, but quantitative assessment of variation among replicated studies at multiple sites is rare. We investigated patterns of space use for 10 populations of two closely related species of prairie grouse: Greater Prairie-Chickens (Tympanuchus cupido) and Lesser Prairie-Chickens (T. pallidicinctus). Prairie chickens require large, intact tracts of native grasslands, and are umbrella species for conservation of prairie ecosystems in North America. We used resource utilization functions to investigate space use by female prairie chickens during the 6-month breeding season from March through August in relation to lek sites, habitat conditions, and anthropogenic development. Our analysis included data from 382 radio-marked individuals across a major portion of the extant range. Our project is a unique opportunity to study comparative space use of prairie chickens, and we employed standardized methods that facilitated direct comparisons across an ecological gradient of study sites. Median home range size of females varied ~10-fold across 10 sites (3.6–36.7 km2), and home ranges tended to be larger at sites with higher annual precipitation. Proximity to lek sites was a strong and consistent predictor of space use for female prairie chickens at all 10 sites. The relative importance of other predictors of space use varied among sites, indicating that generalized habitat management guidelines may not be appropriate for these two species. Prairie chickens actively selected for prairie habitats, even at sites where ~90% of the land cover within the study area was prairie. A majority of the females monitored in our study (>95%) had activity centers within 5 km of leks, suggesting that conservation efforts can be effectively concentrated near active lek sites. Our data on female space use suggest that lek surveys of male prairie chickens can indirectly assess habitat suitability for females during the breeding season. Lek monitoring and surveys for new leks provide information on population trends, but can also guide management actions aimed at improving nesting and brood-rearing habitats.

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

PubMed

Heng, Sabrina; Stieglitz, Kimberly A; Eldo, Joby; Xia, Jiarong; Cardia, James P; Kantrowitz, Evan R

2006-08-22

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.

Concentration and mindfulness meditations: unique forms of consciousness?

PubMed

Dunn, B R; Hartigan, J A; Mikulas, W L

1999-09-01

Electroencephalographic (EEG) recordings from 19 scalp recording sites were used to differentiate among two posited unique forms of mediation, concentration and mindfulness, and a normal relaxation control condition. Analyzes of all traditional frequency bandwidth data (i.e., delta 1-3 Hz; theta, 4-7 Hz; alpha, 8-12 Hz; beta 1, 13-25 Hz; beta 2, 26-32 Hz) showed strong mean amplitude frequency differences between the two meditation conditions and relaxation over numerous cortical sites. Furthermore, significant differences were obtained between concentration and mindfulness states at all bandwidths. Taken together, our results suggest that concentration and mindfulness "meditations" may be unique forms of consciousness and are not merely degrees of a state of relaxation.

Evaluation of soil contamination by explosives and metals at the Land Force Central Area Training Centre (LFCA TC) Meaford, Ontario (Phase I)

DTIC Science & Technology

2009-05-01

found in ammunition. The site characterization allowed the development of a unique expertise and positioned our departments to better understand the...environnementaux des composés énergétiques que l’on retrouve dans les munitions. La caractérisation des sites a permis de développer une expertise unique et a...éventualités pour prendre des mesures correctives, si nécessaire. Les premiers sites d’entraînement à être évalués ont été des bases de l’armée, telles que

On the quantitative inventory of the riverscape

USGS Publications Warehouse

Leopold, Luna Bergere; O'Brien Marchand, Maura

1968-01-01

In the vicinity of Berkeley, California, 24 minor valleys were described in terms of factors chosen to represent aspects of the river landscape. A total of 28 factors were evaluated at each site. Some were directly measurable, others were estimated, but each observation was assigned to one of five categories for that factor. Each factor for each site was then expressed as a uniqueness ratio, which depended on the number of sites being in the same category. The uniqueness ratio is believed to represent one way the scarcity of a given riverscape can be ranked quantitatively without bias based on notions of good or bad, and without assigning monetary value.

Synthesis, characterization and photocatalytic performance of chemically exfoliated MoS2

NASA Astrophysics Data System (ADS)

Prabhakar Vattikuti, S. V.; Shim, Jaesool

2018-03-01

Two-dimensional (2D) layered structure transition metal dichalcogenides (TMDs) has gained huge attention and importance for photocatalytic energy conversion because of their unique properties. Molybdenum disulfide (MoS2) nanosheets were synthesized via one-pot method and exfoliated in (dimethylformamide) DMF solution. Subsequent exfoliated MoS2 nanosheets (e-MoS2) were used as photocatalysts for degradation of Rhodamine B (RhB) pollutant under solar light irradiation. The e-MoS2 nanosheets exhibited excellent photocatalytic activity than that of pristine MoS2, owing to high specific surface area with enormous active sites and light absorption capacity. In addition, e-MoS2 demonstrated remarkable photocatalytic stability.

Crystal structure of the C-terminal SH3 domain of the adaptor protein GADS in complex with SLP-76 motif peptide reveals a unique SH3-SH3 interaction.

PubMed

Dimasi, Nazzareno

2007-01-01

The Grb2-like adaptor protein GADS is essential for tyrosine kinase-dependent signaling in T lymphocytes. Following T cell receptor ligation, GADS interacts through its C-terminal SH3 domain with the adaptors SLP-76 and LAT, to form a multiprotein signaling complex that is crucial for T cell activation. To understand the structural basis for the selective recognition of GADS by SLP-76, herein is reported the crystal structure at 1.54 Angstrom of the C-terminal SH3 domain of GADS bound to the SLP-76 motif 233-PSIDRSTKP-241, which represents the minimal binding site. In addition to the unique structural features adopted by the bound SLP-76 peptide, the complex structure reveals a unique SH3-SH3 interaction. This homophilic interaction, which is observed in presence of the SLP-76 peptide and is present in solution, extends our understanding of the molecular mechanisms that could be employed by modular proteins to increase their signaling transduction specificity.

Hourly elemental concentrations in PM2.5 aerosols sampled simultaneously at urban background and road site

NASA Astrophysics Data System (ADS)

Dall'Osto, M.; Querol, X.; Amato, F.; Karanasiou, A.; Lucarelli, F.; Nava, S.; Calzolai, G.; Chiari, M.

2012-08-01

Hourly-resolved aerosol chemical speciation data can be a highly powerful tool to determine the source origin of atmospheric pollutants in urban Environments. Aerosol mass concentrations of seventeen elements (Na, Mg, Al, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Sr and Pb) were obtained by time (1 h) and size (PM2.5 particulate matter <2.5 μm) resolved Particle Induced X-ray Emission (PIXE) measurements. In the Marie Curie FP7-EU framework of SAPUSS (Solving Aerosol Problems by Using Synergistic Strategies), the unique approach used is the simultaneous PIXE measurements at two monitoring sites: urban background (UB) and a street canyon traffic road site (RS). Elements related to primary non exhaust traffic emission (Fe, Cu), dust resuspension (Ca) and anthropogenic Cl were found enhanced at the RS, whereas industrial related trace metals (Zn, Pb, Mn) were found at higher concentrations at the more ventilated UB site. When receptor modelling was performed with positive matrix factorization (PMF), nine different aerosol sources were identified at both sites: three types of regional aerosols (secondary sulphate (S) - 27%, biomass burning (K) - 5%, sea salt (Na-Mg) - 17%), three types of dust aerosols (soil dust (Al-Ti) - 17%, urban crustal dust (Ca) - 6%, and primary traffic non exhaust brake dust (Fe-Cu) - 7%), and three types industrial aerosol plumes-like events (shipping oil combustion (V-Ni) - 17%, industrial smelters (Zn-Mn) - 3%, and industrial combustion (Pb-Cl) - 5%). The validity of the PMF solution of the PIXE data is supported by strong correlations with external single particle mass spectrometry measurements. Beside apportioning the aerosol sources, some important air quality related conclusions can be drawn about the PM2.5 fraction simultaneously measured at the UB and RS sites: (1) the regional aerosol sources impact both monitoring sites at similar concentrations regardless their different ventilation conditions; (2) by contrast, local industrial aerosol plumes associated with shipping oil combustion and smelters activities have a higher impact on the more ventilated UB site; (3) a unique source of Pb-Cl (associated with industrial combustion emissions) is found a to be the major (82%) source of Cl in the urban agglomerate; (4) PM2.5 traffic brake dust (Fe-Cu) is mainly primarily emitted and not resuspended, whereas PM2.5 urban crustal dust (Ca) is found mainly resuspended by both traffic vortex and sea breeze; (5) urban dust (Ca) is found the aerosol source most affected by land wetness, reduced by a factor of eight during rainy days and suggesting that wet roads may be a solution for reducing dust concentrations in road sites, far more effective than street sweeping activities.

GetHealthyHarlem.org: Developing a Web Platform for Health Promotion and Wellness Driven by and for the Harlem Community

PubMed Central

Khan, Sharib A.; Ancker, Jessica S.; Li, Jianhua; Kaufman, David; Hutchinson, Carly; Cohall, Alwyn; Kukafka, Rita

2009-01-01

GetHealthyHarlem.org is a community website developed on an open-source platform to facilitate collaborative development of health content through participatory action research (PAR) principles. The website was developed to enable the Harlem community to create a shared health and wellness knowledgebase, to enable discourse about local and culturally relevant health information, and to foster social connections between community members and health promotion organizations. The site is gaining active use with more than 9,500 unique site visits in the six months since going live in November, 2008. In ongoing research studies, we are using the website to explore how the PAR model can be applied to the development of a community health website. PMID:20351872

Boronic acid-containing CXCR1/2 antagonists: optimization of metabolic stability, in vivo evaluation, and a proposed receptor binding model

PubMed Central

Maeda, Dean Y.; Peck, Angela M.; Schuler, Aaron D.; Quinn, Mark T.; Kirpotina, Liliya N.; Wicomb, Winston N.; Auten, Richard L.; Gundla, Rambabu; Zebala, John A.

2015-01-01

Blockade of undesired neutrophil migration to sites of inflammation remains an area of substantial pharmaceutical interest. To effect this blockade, a validated therapeutic target is antagonism of the chemokine receptor CXCR2. Herein we report the discovery of 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide 6, an antagonist with activity at both CXCR1 and CXCR2 receptors (IC50 values 31 and 21 nM, respectively). Compound 6 exhibited potent inhibition of neutrophil influx in a rat model of pulmonary inflammation, and is hypothesized to interact with a unique intracellular binding site on CXCR2. Compound 6 (SX-576) is undergoing further investigation as a potential therapy for pulmonary inflammation. PMID:25933594

Synthesis, bioactivity, 3D-QSAR studies of novel dibenzofuran derivatives as PTP-MEG2 inhibitors

PubMed Central

Zhang, Yu-Ze; Jin, Wen-Yan; Li, Hong-Lian; Zhou, Hui; Cheng, Xian-Chao; Wang, Run-Ling

2017-01-01

PTP-MEG2 plays a critical role in the diverse cell signalling processes, so targeting PTP-MEG2 is a promising strategy for various human diseases treatments. In this study, a series of novel dibenzofuran derivatives was synthesized and assayed for their PTP-MEG2 inhibitory activities. 10a with highest inhibitory activity (320 nM) exhibited significant selectivity for PTP-MEG2 over its close homolog SHP2, CDC25 (IC50 > 50 μM). By means of the powerful “HipHop” technique, a 3D-QSAR study was carried out to explore structure activity relationship of these molecules. The generated pharmacophore model revealed that the one RA, three Hyd, and two HBA features play an important role in binding to the active site of the target protein-PTP-MEG2. Docking simulation study indicated that 10a achieved its potency and specificity for PTP-MEG2 by targeting unique nearby peripheral binding pockets and the active site. The absorption, distribution, metabolism and excretion (ADME) predictions showed that the 11 compounds hold high potential to be novel lead compounds for targeting PTP-MEG2. Our findings here can provide a new strategy or useful insights for designing the effective PTP-MEG2 inhibitors. PMID:28388567

Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein*

PubMed Central

Sawano, Yoriko; Hatano, Ken-ichi; Miyakawa, Takuya; Tanokura, Masaru

2008-01-01

Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed φ dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition. PMID:18948264

Understanding fibroblast activation protein (FAP): substrates, activities, expression and targeting for cancer therapy.

PubMed

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.

Specialized rules of gene transcription in male germ cells: the CREM paradigm.

PubMed

Monaco, Lucia; Kotaja, Noora; Fienga, Giulia; Hogeveen, Kevin; Kolthur, Ullas S; Kimmins, Sarah; Brancorsini, Stefano; Macho, Betina; Sassone-Corsi, Paolo

2004-12-01

Specialized transcription complexes that coordinate the differentiation programme of spermatogenesis have been found in germ cells, which display specific differences in the components of the general transcription machinery. The TATA-binding protein family and its associated cofactors, for example, show upregulated expression in testis. In this physiological context, transcriptional control mediated by the activator cAMP response element modulator (CREM) represents an established paradigm. Somatic cell activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CREB-binding protein. In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, activator of CREM in the testis (ACT), which confers a powerful, phosphorylation-independent activation capacity. The function of ACT was found to be regulated by the testis-specific kinesin KIF17b. Here we discuss some aspects of the testis-specific transcription machinery, whose function is essential for the process of spermatogenesis.

Unique binding behavior of the recently approved angiotensin II receptor blocker azilsartan compared with that of candesartan

PubMed Central

Miura, Shin-ichiro; Okabe, Atsutoshi; Matsuo, Yoshino; Karnik, Sadashiva S; Saku, Keijiro

2014-01-01

The angiotensin II type 1 (AT1) receptor blocker (ARB) candesartan strongly reduces blood pressure (BP) in patients with hypertension and has been shown to have cardioprotective effects. A new ARB, azilsartan, was recently approved and has been shown to provide a more potent 24-h sustained antihypertensive effect than candesartan. However, the molecular interactions of azilsartan with the AT1 receptor that could explain its strong BP-lowering activity are not yet clear. To address this issue, we examined the binding affinities of ARBs for the AT1 receptor and their inverse agonist activity toward the production of inositol phosphate (IP), and we constructed docking models for the interactions between ARBs and the receptor. Azilsartan, unlike candesartan, has a unique moiety, a 5-oxo-1,2,4-oxadiazole, in place of a tetrazole ring. Although the results regarding the binding affinities of azilsartan and candesartan demonstrated that these ARBs interact with the same sites in the AT1 receptor (Tyr113, Lys199 and Gln257), the hydrogen bonding between the oxadiazole of azilsartan-Gln257 is stronger than that between the tetrazole of candesartan-Gln257, according to molecular docking models. An examination of the inhibition of IP production by ARBs using constitutively active mutant receptors indicated that inverse agonist activity required azilsartan–Gln257 interaction and that azilsartan had a stronger interaction with Gln257 than candesartan. Thus, we speculate that azilsartan has a unique binding behavior to the AT1 receptor due to its 5-oxo-1,2,4-oxadiazole moiety and induces stronger inverse agonism. This property of azilsartan may underlie its previously demonstrated superior BP-lowering efficacy compared with candesartan and other ARBs. PMID:23034464

Unique binding behavior of the recently approved angiotensin II receptor blocker azilsartan compared with that of candesartan.

PubMed

Miura, Shin-ichiro; Okabe, Atsutoshi; Matsuo, Yoshino; Karnik, Sadashiva S; Saku, Keijiro

2013-02-01

The angiotensin II type 1 (AT(1)) receptor blocker (ARB) candesartan strongly reduces blood pressure (BP) in patients with hypertension and has been shown to have cardioprotective effects. A new ARB, azilsartan, was recently approved and has been shown to provide a more potent 24-h sustained antihypertensive effect than candesartan. However, the molecular interactions of azilsartan with the AT(1) receptor that could explain its strong BP-lowering activity are not yet clear. To address this issue, we examined the binding affinities of ARBs for the AT(1) receptor and their inverse agonist activity toward the production of inositol phosphate (IP), and we constructed docking models for the interactions between ARBs and the receptor. Azilsartan, unlike candesartan, has a unique moiety, a 5-oxo-1,2,4-oxadiazole, in place of a tetrazole ring. Although the results regarding the binding affinities of azilsartan and candesartan demonstrated that these ARBs interact with the same sites in the AT(1) receptor (Tyr(113), Lys(199) and Gln(257)), the hydrogen bonding between the oxadiazole of azilsartan-Gln(257) is stronger than that between the tetrazole of candesartan-Gln(257), according to molecular docking models. An examination of the inhibition of IP production by ARBs using constitutively active mutant receptors indicated that inverse agonist activity required azilsartan-Gln(257) interaction and that azilsartan had a stronger interaction with Gln(257) than candesartan. Thus, we speculate that azilsartan has a unique binding behavior to the AT(1) receptor due to its 5-oxo-1,2,4-oxadiazole moiety and induces stronger inverse agonism. This property of azilsartan may underlie its previously demonstrated superior BP-lowering efficacy compared with candesartan and other ARBs.

Pericyclic reactions catalyzed by chorismate-utilizing enzymes

PubMed Central

Lamb, Audrey L.

2011-01-01

One of the fundamental questions of enzymology is how catalytic power is derived. This review focuses on recent developments in the structure-function relationships of chorismate-utilizing enzymes involved in siderophore biosynthesis to provide insight into the biocatalysis of pericyclic reactions. Specifically, salicylate synthesis by the two-enzyme pathway in Pseudomonas aeruginosa is examined. The isochorismate-pyruvate lyase is discussed in the context of its homologues, the chorismate mutases, and the isochorismate synthase is compared to its homologues in the MST-family (menaquinone, siderophore or tryptophan biosynthesis) of enzymes. The tentative conclusion is that the activities observed cannot be reconciled by inspection of the active site participants alone. Instead, individual activities must arise from unique dynamic properties of each enzyme that are tuned to promote specific chemistries. PMID:21823653

Multiple cis-acting elements involved in up-regulation of a cytochrome P450 gene conferring resistance to deltamethrin in smal brown planthopper, Laodelphax striatellus (Fallén).

PubMed

Pu, Jian; Sun, Haina; Wang, Jinda; Wu, Min; Wang, Kangxu; Denholm, Ian; Han, Zhaojun

2016-11-01

As well as arising from single point mutations in binding sites or detoxifying enzymes, it is likely that insecticide resistance mechanisms are frequently controlled by multiple genetic factors, resulting in resistance being inherited as a quantitative trait. However, empirical evidence for this is still rare. Here we analyse the causes of up-regulation of CYP6FU1, a monoxygenase implicated in resistance to deltamethrin in the rice pest Laodelphax striatellus. The 5'-flanking region of this gene was cloned and sequenced from individuals of a susceptible and a resistant strain. A luminescent reporter assay was used to evaluate different 5'-flanking regions and their fragments for promoter activity. Mutations enhancing promoter activity in various fragments were characterized, singly and in combination, by site mutation recovery. Nucleotide diversity in flanking sequences was greatly reduced in deltamethrin-resistant insects compared to susceptible ones. Phylogenetic sequence analysis found that CYP6FU1 had five different types of 5'-flanking region. All five types were present in a susceptible strain but only a single type showing the highest promoter activity was present in a resistant strain. Four cis-acting elements were identified whose influence on up-regulation was much more pronounced in combination than when present singly. Of these, two were new transcription factor (TF) binding sites produced by mutations, another one was also a new TF binding site alternated from an existing one, and the fourth was a unique transcription start site. These results demonstrate that multiple cis-acting elements are involved in up-regulating CYP6FU1 to generate a resistance phenotype. Copyright © 2016 Elsevier Ltd. All rights reserved.

Berberine Induces Toxicity in HeLa Cells through Perturbation of Microtubule Polymerization by Binding to Tubulin at a Unique Site.

PubMed

Raghav, Darpan; Ashraf, Shabeeba M; Mohan, Lakshmi; Rathinasamy, Krishnan

2017-05-23

Berberine has been used traditionally for its diverse pharmacological actions. It exhibits remarkable anticancer activities and is currently under clinical trials. In this study, we report that the anticancer activity of berberine could be partly due to its inhibitory actions on tubulin and microtubule assembly. Berberine inhibited the proliferation of HeLa cells with an IC 50 of 18 μM and induced significant depolymerization of interphase and mitotic microtubules. At its IC 50 , berberine exerted a moderate G2/M arrest and mitotic block as detected by fluorescence-activated cell sorting analysis and fluorescence microscopy, respectively. In a wound closure assay, berberine inhibited the migration of HeLa cells at concentrations lower than its IC 50 , indicating its excellent potential as an anticancer agent. In vitro studies with tubulin isolated from goat brain indicated that berberine binds to tubulin at a single site with a K d of 11 μM. Berberine inhibited the assembly of tubulin into microtubules and also disrupted the preformed microtubules polymerized in the presence of glutamate and paclitaxel. Competition experiments indicated that berberine could partially displace colchicine from its binding site. Results from fluorescence resonance energy transfer, computational docking, and molecular dynamics simulations suggest that berberine forms a stable complex with tubulin and binds at a novel site 24 Å from the colchicine site on the β-tubulin. Data obtained from synchronous fluorescence analysis of the tryptophan residues of tubulin and from the Fourier transform infrared spectroscopy studies revealed that binding of berberine alters the conformation of the tubulin heterodimer, which could be the molecular mechanism behind the depolymerizing effects on tubulin assembly.

Specificity of Processing α-glucosidase I is guided by the substrate conformation: crystallographic and in silico studies.

PubMed

Barker, Megan K; Rose, David R

2013-05-10

The enzyme “GluI” is key to the synthesis of critical glycoproteins in the cell. We have determined the structure of GluI, and modeled binding with its unique sugar substrate. The specificity of this interaction derives from a unique conformation of the substrate. Understanding the mechanism of the enzyme is of basic importance and relevant to potential development of antiviral inhibitors. Processing α-glucosidase I (GluI) is a key member of the eukaryotic N-glycosylation processing pathway, selectively catalyzing the first glycoprotein trimming step in the endoplasmic reticulum. Inhibition of GluI activity impacts the infectivity of enveloped viruses; however, despite interest in this protein from a structural, enzymatic, and therapeutic standpoint, little is known about its structure and enzymatic mechanism in catalysis of the unique glycan substrate Glc3Man9GlcNAc2. The first structural model of eukaryotic GluI is here presented at 2-Å resolution. Two catalytic residues are proposed, mutations of which result in catalytically inactive, properly folded protein. Using Autodocking methods with the known substrate and inhibitors as ligands, including a novel inhibitor characterized in this work, the active site of GluI was mapped. From these results, a model of substrate binding has been formulated, which is most likely conserved in mammalian GluI.

Archaeology of Void Spaces

NASA Astrophysics Data System (ADS)

Look, Cory

The overall goal of this research is to evaluate the efficacy of pXRF for the identification of ancient activity areas at Pre-Columbian sites in Antigua that range across time periods, geographic regions, site types with a variety of features, and various states of preservation. These findings have important implications for identifying and reconstructing places full of human activity but void of material remains. A synthesis for an archaeology of void spaces requires the construction of new ways of testing anthrosols, and identifying elemental patterns that can be used to connect people with their places and objects. This research begins with an exploration of rich middens in order to study void spaces. Midden archaeology has been a central focus in Caribbean research, and consists of an accumulation of discarded remnants from past human activities that can be tested against anthrosols. The archaeological collections visited for this research project involved creating new databases to generate a comprehensive inventory of sites, materials excavated, and assemblages available for study. Of the more than 129 Pre-Columbian sites documented in Antigua, few sites have been thoroughly surveyed or excavated. Twelve Pre-Columbian sites, consisting of thirty-six excavated units were selected for study; all of which contained complete assemblages for comparison and soil samples for testing. These excavations consisted almost entirely of midden excavations, requiring new archaeological investigations to be carried out in spaces primarily void of material remains but within the village context. Over the course of three seasons excavations, shovel test pits, and soil augers were used to obtain a variety of anthrosols and archaeological assemblages in order to generate new datasets to study Pre-Columbian activity areas. The selection of two primary case study sites were used for comparison: Indian Creek and Doigs. Findings from this research indicate that accounting for the variety of activity areas that make up a site can imbue a site with an identity of purpose and shed light on how different sites may have served different purposes within a regional framework. Excavations at the site of Indian Creek identified a series of raised middens that enclosed an open space for approximately 1500 years. This research explores this open space, and questions the meaning of 'void' and 'empty' with respect to past human activities. While archaeologists recognize that areas void of material remains are certainly part of the larger site, the question remains, without an understand of these spaces; what aspects of past life are we possibly masking? The integration of anthrosols alongside archaeological excavations and spatial analysis indicate that the site of Indian Creek contained a ceremonial plaza that formed early on and was maintained until abandonment. The spatial distribution of material objects combined with anthrosol studies provided additional evidence of ritual deposits concentrated in one part of the plaza associated with a nearby creek-bed. The second site, Doigs represents one of the last intact undisturbed Early Ceramic Age site of its kind in the Eastern Caribbean. Since its discovery in the 1970's, Doig's has been partially surveyed and excavated. The identification of residential activity areas including several potential structures, bead manufacturing loci, and cooking hearths were used to help test chemical signatures with archaeologically defined activity areas. Findings from this site illustrated the uniqueness of elemental patterns associated with activity areas, and also generated new questions regarding void spaces enriched with elemental patterns associated with concentrations of plant and vegetation debris. It is the hope of this study to contribute to our general knowledge for the identification of ancient activity areas as well as the different places that give sites their identity. These assemblages of activity areas can provide Caribbeanists with an alternative approach to studying social organization at a village scale and generate new discussions regarding island wide-community relationships.

CLIP-seq analysis of multi-mapped reads discovers novel functional RNA regulatory sites in the human transcriptome.

PubMed

Zhang, Zijun; Xing, Yi

2017-09-19

Crosslinking or RNA immunoprecipitation followed by sequencing (CLIP-seq or RIP-seq) allows transcriptome-wide discovery of RNA regulatory sites. As CLIP-seq/RIP-seq reads are short, existing computational tools focus on uniquely mapped reads, while reads mapped to multiple loci are discarded. We present CLAM (CLIP-seq Analysis of Multi-mapped reads). CLAM uses an expectation-maximization algorithm to assign multi-mapped reads and calls peaks combining uniquely and multi-mapped reads. To demonstrate the utility of CLAM, we applied it to a wide range of public CLIP-seq/RIP-seq datasets involving numerous splicing factors, microRNAs and m6A RNA methylation. CLAM recovered a large number of novel RNA regulatory sites inaccessible by uniquely mapped reads. The functional significance of these sites was demonstrated by consensus motif patterns and association with alternative splicing (splicing factors), transcript abundance (AGO2) and mRNA half-life (m6A). CLAM provides a useful tool to discover novel protein-RNA interactions and RNA modification sites from CLIP-seq and RIP-seq data, and reveals the significant contribution of repetitive elements to the RNA regulatory landscape of the human transcriptome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification and Characterization of a Novel Member of the Radical AdoMet Enzyme Superfamily and Implications for the Biosynthesis of the Hmd Hydrogenase Active Site Cofactor▿ †

PubMed Central

McGlynn, Shawn E.; Boyd, Eric S.; Shepard, Eric M.; Lange, Rachel K.; Gerlach, Robin; Broderick, Joan B.; Peters, John W.

2010-01-01

The genetic context, phylogeny, and biochemistry of a gene flanking the H2-forming methylene-H4-methanopterin dehydrogenase gene (hmdA), here designated hmdB, indicate that it is a new member of the radical S-adenosylmethionine enzyme superfamily. In contrast to the characteristic CX3CX2C or CX2CX4C motif defining this family, HmdB contains a unique CX5CX2C motif. PMID:19897660

Crystal structure of casein kinase-1, a phosphate-directed protein kinase.

PubMed Central

Xu, R M; Carmel, G; Sweet, R M; Kuret, J; Cheng, X

1995-01-01

The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate. Images PMID:7889932

The inner workings of the hydrazine synthase multiprotein complex.

PubMed

Dietl, Andreas; Ferousi, Christina; Maalcke, Wouter J; Menzel, Andreas; de Vries, Simon; Keltjens, Jan T; Jetten, Mike S M; Kartal, Boran; Barends, Thomas R M

2015-11-19

Anaerobic ammonium oxidation (anammox) has a major role in the Earth's nitrogen cycle and is used in energy-efficient wastewater treatment. This bacterial process combines nitrite and ammonium to form dinitrogen (N2) gas, and has been estimated to synthesize up to 50% of the dinitrogen gas emitted into our atmosphere from the oceans. Strikingly, the anammox process relies on the highly unusual, extremely reactive intermediate hydrazine, a compound also used as a rocket fuel because of its high reducing power. So far, the enzymatic mechanism by which hydrazine is synthesized is unknown. Here we report the 2.7 Å resolution crystal structure, as well as biophysical and spectroscopic studies, of a hydrazine synthase multiprotein complex isolated from the anammox organism Kuenenia stuttgartiensis. The structure shows an elongated dimer of heterotrimers, each of which has two unique c-type haem-containing active sites, as well as an interaction point for a redox partner. Furthermore, a system of tunnels connects these active sites. The crystal structure implies a two-step mechanism for hydrazine synthesis: a three-electron reduction of nitric oxide to hydroxylamine at the active site of the γ-subunit and its subsequent condensation with ammonia, yielding hydrazine in the active centre of the α-subunit. Our results provide the first, to our knowledge, detailed structural insight into the mechanism of biological hydrazine synthesis, which is of major significance for our understanding of the conversion of nitrogenous compounds in nature.

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

PubMed

Strotmeier, Jasmin; Gu, Shenyan; Jutzi, Stephan; Mahrhold, Stefan; Zhou, Jie; Pich, Andreas; Eichner, Timo; Bigalke, Hans; Rummel, Andreas; Jin, Rongsheng; Binz, Thomas

2011-07-01

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins. © 2011 Blackwell Publishing Ltd.

Planktonic food web structure at a coastal time-series site: II. Spatiotemporal variability of microbial trophic activities

NASA Astrophysics Data System (ADS)

Connell, Paige E.; Campbell, Victoria; Gellene, Alyssa G.; Hu, Sarah K.; Caron, David A.

2017-03-01

The grazing activities of phagotrophic protists on various microbial assemblages play key roles in determining the amount of carbon available for higher trophic levels and for export out of the photic zone. However, comparisons of the proportion of carbon consumed from the phytoplankton (cyanobacteria+photosynthetic eukaryotes) and heterotrophic bacteria (bacteria+archaea, excluding cyanobacteria) are rare. In this study, microbial community composition, phytoplankton growth and mortality rates (total chlorophyll a, Synechococcus, Prochlorococcus, and photosynthetic picoeukaryotes), and bacterial mortality rates were measured seasonally from 2012 to 2014 in the surface waters of three environmentally distinct sites in the San Pedro Channel, off the coast of southern CA, USA. Higher nutrient concentrations at the nearshore site supported community standing stocks that were 1.3-4.5x those found offshore, yet average growth and grazing rates of the phytoplankton and bacterial assemblages were generally similar between sites and across seasons. Thus, the amount of carbon consumed by the grazer assemblage was largely dictated by prey standing stocks. Heterotrophic bacteria constituted an important source of carbon for microbial consumers, particularly at the two offshore sites where bacterial carbon consumed was roughly equivalent to the amount of phytoplankton carbon consumed. Carbon removal by grazers at the nearshore station was predominantly from the diatoms, which were the primary component of the photosynthetic community at that site. This study highlights the significant contribution of protistan-bacterial trophic interactions to planktonic food webs and provides unique community composition and turnover data to inform biogeochemical models.

Telemetry-Determined Habitat Use Informs Multi-Species Habitat Management in an Urban Harbour

NASA Astrophysics Data System (ADS)

Rous, Andrew M.; Midwood, Jonathon D.; Gutowsky, Lee F. G.; Lapointe, Nicolas W. R.; Portiss, Rick; Sciscione, Thomas; Wells, Mathew G.; Doka, Susan E.; Cooke, Steven J.

2017-01-01

Widespread human development has led to impairment of freshwater coastal wetlands and embayments, which provide critical and unique habitat for many freshwater fish species. This is particularly evident in the Laurentian Great Lakes, where such habitats have been severely altered over the last century as a result of industrial activities, urbanization, dredging and infilling. In Toronto Harbour, extensive restoration efforts have been directed towards improving the amount and quality of aquatic habitat, especially for fishes. To evaluate the effectiveness of this restoration work, use of the restored area by both target species and the fish community as a whole must be assessed. Individuals from four species (Common Carp, Largemouth Bass, Northern Pike and Yellow Perch) were tagged and tracked continuously for 1 year using an acoustic telemetry array in Toronto Harbour area of Lake Ontario. Daily site fidelity was estimated using a mixed-effects logistic regression model. Daily site fidelity was influenced by habitat restoration and its interactions with species and body size, as well as season and its interactions with species and body size. Daily site fidelity was higher in restored sites compared to non-restored sites for Yellow Perch and Northern Pike, but lower for Largemouth Bass and Common Carp. For all species, daily site fidelity estimates were highest during the summer and lowest during autumn. The approach used here has merit for evaluating restoration success and informing future habitat management activities. Creating diverse habitats that serve multiple functions and species are more desirable than single-function-oriented or single-species-oriented designs.

Telemetry-Determined Habitat Use Informs Multi-Species Habitat Management in an Urban Harbour.

PubMed

Rous, Andrew M; Midwood, Jonathon D; Gutowsky, Lee F G; Lapointe, Nicolas W R; Portiss, Rick; Sciscione, Thomas; Wells, Mathew G; Doka, Susan E; Cooke, Steven J

2017-01-01

Widespread human development has led to impairment of freshwater coastal wetlands and embayments, which provide critical and unique habitat for many freshwater fish species. This is particularly evident in the Laurentian Great Lakes, where such habitats have been severely altered over the last century as a result of industrial activities, urbanization, dredging and infilling. In Toronto Harbour, extensive restoration efforts have been directed towards improving the amount and quality of aquatic habitat, especially for fishes. To evaluate the effectiveness of this restoration work, use of the restored area by both target species and the fish community as a whole must be assessed. Individuals from four species (Common Carp, Largemouth Bass, Northern Pike and Yellow Perch) were tagged and tracked continuously for 1 year using an acoustic telemetry array in Toronto Harbour area of Lake Ontario. Daily site fidelity was estimated using a mixed-effects logistic regression model. Daily site fidelity was influenced by habitat restoration and its interactions with species and body size, as well as season and its interactions with species and body size. Daily site fidelity was higher in restored sites compared to non-restored sites for Yellow Perch and Northern Pike, but lower for Largemouth Bass and Common Carp. For all species, daily site fidelity estimates were highest during the summer and lowest during autumn. The approach used here has merit for evaluating restoration success and informing future habitat management activities. Creating diverse habitats that serve multiple functions and species are more desirable than single-function-oriented or single-species-oriented designs.

The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases*

PubMed Central

Peterson, Ryan L.; Galaleldeen, Ahmad; Villarreal, Johanna; Taylor, Alexander B.; Cabelli, Diane E.; Hart, P. John; Culotta, Valeria C.

2016-01-01

In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. Recently, we reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. Despite these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or “pseudofungi” species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies. PMID:27535222

Structures of a bifunctional cell wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 DL-endopeptidase.

PubMed

Xu, Qingping; Chiu, Hsiu-Ju; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Lesley, Scott A; Godzik, Adam; Elsliger, Marc-André; Deacon, Ashley M; Wilson, Ian A

2014-01-09

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state). © 2013.

The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases

DOE PAGES

Peterson, Ryan L.; Galaleldeen, Ahmad; Villarreal, Johanna; ...

2016-08-17

In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. We reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. In spite of these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or “pseudofungi” species. It is the only form ofmore » extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. Furthermore, SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies.« less

Field measures show methanotroph sensitivity to soil moisture follows precipitation regime of the grassland sites across the US Great Plains

NASA Astrophysics Data System (ADS)

Koyama, A.; Webb, C. T.; Johnson, N. G.; Brewer, P. E.; von Fischer, J. C.

2015-12-01

Methane uptake rates are known to have temporal variation in response to changing soil moisture levels. However, the relative importance of soil diffusivity vs. methanotroph physiology has not been disentangled to date. Testing methanotroph physiology in the laboratory can lead to misleading results due to changes in the fine-scale habitat where methanotrophs reside. To assay the soil moisture sensitivity of methanotrophs under field conditions, we studied 22 field plots scattered across eight Great Plains grassland sites that differed in precipitation regime and soil moisture, making ca. bi-weekly measures during the growing seasons over three years. Quantification of methanotroph activity was achieved from chamber-based measures of methane uptake coincident with SF6-derived soil diffusivity, and interpretation in a reaction-diffusion model. At each plot, we also measured soil water content (SWC), soil temperature and inorganic nitrogen (N) contents. We also assessed methanotroph community composition via 454 sequencing of the pmoA gene. Statistical analyses showed that methanotroph activity had a parabolic response with SWC (concave down), and significant differences in the shape of this response among sites. Moreover, we found that the SWC at peak methanotroph activity was strongly correlated with mean annual precipitation (MAP) of the site. The sequence data revealed distinct composition patterns, with structure that was associated with variation in MAP and soil texture. These results suggest that local precipitation regime shapes methanotroph community composition, which in turn lead to unique sensitivity of methane uptake rates with soil moisture. Our findings suggest that methanotroph activity may be more accurately modeled when the biological and environmental responses are explicitly described.

Changes in deep-sea carbonate-hosted microbial communities associated with high and low methane flux

NASA Astrophysics Data System (ADS)

Case, D. H.; Steele, J. A.; Chadwick, G.; Mendoza, G. F.; Levin, L. A.; Orphan, V. J.

2012-12-01

Methane seeps on continental shelves are rich in authigenic carbonates built of methane-derived carbon. These authigenic carbonates are home to micro- and macroscopic communities whose compositions are thus far poorly constrained but are known to broadly depend on local methane flux. The formation of authigenic carbonates is itself a result of microbial metabolic activity, as associations of anaerobic methane oxidizing archaea (ANME) and sulfate reducing bacteria (SRB) in the sediment subsurface increase both dissolved inorganic carbon (DIC) and alkalinity in pore waters. This 1:1 increase in DIC and alkalinity promotes the precipitation of authigenic carbonates. In this study, we performed in situ manipulations to test the response of micro- and macrofaunal communities to a change in methane flux. Methane-derived authigenic carbonates from two locations at Hydrate Ridge, OR, USA (depth range 595-604 mbsl), were transplanted from "active" cold seep sites (high methane flux) to "inactive" background sites (low methane flux), and vise versa, for one year. Community diversity surveys using T-RFLP and 16S rRNA clone libraries revealed how both bacterial and archaeal assemblages respond to this change in local environment, specifically demonstrating reproducible shifts in different ANME groups (ANME-1 vs. ANME-2). Animal assemblage composition also shifted during transplantation; gastropod representation increased (relative to control rocks) when substrates were moved from inactive to active sites and polychaete, crustacean and echinoderm representation increased when substrates were moved from active to inactive sites. Combined with organic and inorganic carbon δ13C measurements and mineralogy, this unique in situ experiment demonstrates that authigenic carbonates are viable habitats, hosting microbial and macrofaunal communities capable of responding to changes in external environment over relatively short time periods.

Visualizing Active-Site Dynamics in Single Crystals of HePTP: Opening of the WPD Loop Involves Coordinated Movement of the E Loop

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

D Critton; L Tautz; R Page

2011-12-31

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loopmore » in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an 'atypically open' conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.« less

Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures.

PubMed

Jain, Kanishk; Warmack, Rebeccah A; Debler, Erik W; Hadjikyriacou, Andrea; Stavropoulos, Peter; Clarke, Steven G

2016-08-26

In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

CYP2C9 Amino Acid Residues Influencing Phenytoin Turnover and Metabolite Regio- and Stereochemistry

PubMed Central

Mosher, Carrie M.; Tai, Guoying; Rettie, Allan E.

2009-01-01

Phenytoin has been an effective anticonvulsant agent for over 60 years, although its clinical use is complicated by nonlinear pharmacokinetics, a narrow therapeutic index, and metabolically based drug-drug interactions. Although it is well established that CYP2C9 is the major cytochrome P450 enzyme controlling metabolic elimination of phenytoin through its oxidative conversion to (S)-5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), nothing is known about the amino acid binding determinants within the CYP2C9 active site that promote metabolism and maintain the tight stereocontrol of hydroxy metabolite formation. This knowledge gap was addressed here through the construction of nine active site mutants at amino acid positions Phe100, Arg108, Phe114, Leu208, and Phe476 and in vitro analysis of the steady-state kinetics and stereochemistry of p-HPPH formation. The F100L and F114W mutants exhibited 4- to 5-fold increases in catalytic efficiency, whereas the F100W, F114L, F476L, and F476W mutants lost >90% of their phenytoin hydroxylation capacity. This pattern of effects differs substantially from that found previously for (S)-warfarin and (S)-flurbiprofen metabolism, suggesting that these three ligands bind within discrete locations in the CYP2C9 active site. Only the F114L, F476L, and L208V mutants altered phenytoin's orientation during catalytic turnover. The L208V mutant also uniquely demonstrated enhanced 6-hydroxylation of (S)-warfarin. These latter data provide the first experimental evidence for a role of the F-G loop region in dictating the catalytic orientation of substrates within the CYP2C9 active site. PMID:19258521

Status of Environmental Management Initiatives to Accelerate the Reduction of Environmental Risks and Challenges Posed by the Legacy of the Cold War

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

None

2009-01-01

Fifty years of nuclear weapons production and energy research in the United States during the Cold War generated large amounts of radioactive wastes, spent nuclear fuel (SNF), excess plutonium and uranium, thousands of contaminated facilities, and contaminated soil and groundwater. During most of that half century, the Nation did not have the environmental regulatory structure or nuclear waste cleanup technologies that exist today. The result was a legacy of nuclear waste that was stored and disposed of in ways now considered unacceptable. Cleaning up and ultimately disposing of these wastes is the responsibility of the U.S. Department of Energy (DOE).more » In 1989, DOE established the Office of Environmental Management (EM) to solve the large scale and technically challenging risks posed by the world's largest nuclear cleanup. This required EM to build a new nuclear cleanup infrastructure, assemble and train a technically specialized workforce, and develop the technologies and tools required to safely decontaminate, disassemble, stabilize, disposition, and remediate unique radiation hazards. The sites where nuclear activities produced legacy waste and contamination include the original Manhattan Project sites--Los Alamos, New Mexico; Hanford, Washington; and Oak Ridge, Tennessee--as well as major Cold War sites, such as Savannah River Site, South Carolina; the Idaho National Laboratory, Idaho; Rocky Flats Plant, Colorado; and Fernald, Ohio. Today EM has responsibility for nuclear cleanup activities at 21 sites covering more than two million acres in 13 states, and employs more than 30,000 Federal and contractor employees, including scientists, engineers and hazardous waste technicians. This cleanup poses unique, technically complex problems, which must be solved under the most hazardous of conditions, and which will require billions of dollars a year for several more decades. The EM program focus during its first 10 years was on managing the most urgent risks and maintaining safety at each site while negotiating state and Federal environmental compliance agreements. The program also concentrated on characterizing waste and nuclear materials and assessing the magnitude and extent of environmental contamination. By the late 1990s, EM had made significant progress in identifying and characterizing the extent of contamination and cleanup required and began transitioning from primarily a characterization and stabilization program to an active cleanup and closure program. During that time, EM formulated multi-year cleanup and closure plans, which contributed to cleanup progress; however, reducing the overall environmental risk associated with the cleanup program remained a challenge. In response, the Secretary of Energy directed a review of the EM program be undertaken. The resulting 'Top-to Bottom Review' re-directed the program focus from managing risks to accelerating the reduction of these risks.« less

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

PubMed

Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J

2000-08-01

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

The impact of active site mutations of South African HIV PR on drug resistance: Insight from molecular dynamics simulations, binding free energy and per-residue footprints.

PubMed

Ahmed, Shaimaa M; Maguire, Glenn E M; Kruger, Hendrik G; Govender, Thirumala

2014-04-01

Molecular dynamics simulations and binding free energy calculations were used to provide an understanding of the impact of active site drug-resistant mutations of the South African HIV protease subtype C (C-SA HIV PR), V82A and V82F/I84V on drug resistance. Unique per-residue interaction energy 'footprints' were developed to map the overall drug-binding profiles for the wild type and mutants. Results confirmed that these mutations altered the overall binding landscape of the amino acid residues not only in the active site region but also in the flaps as well. Four FDA-approved drugs were investigated in this study; these include ritonavir (RTV), saquinavir (SQV), indinavir (IDV), and nelfinavir (NFV). Computational results compared against experimental findings were found to be complementary. Against the V82F/I84V variant, saquinavir, indinavir, and nelfinavir lose remarkable entropic contributions relative to both wild-type and V82A C-SA HIV PRs. The per-residue energy 'footprints' and the analysis of ligand-receptor interactions for the drug complexes with the wild type and mutants have also highlighted the nature of drug interactions. The data presented in this study will prove useful in the design of more potent inhibitors effective against drug-resistant HIV strains. © 2013 John Wiley & Sons A/S.

Assessing heterogeneity in oligomeric AAA+ machines.

PubMed

Sysoeva, Tatyana A

2017-03-01

ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.

Nuts, nut cracking, and pitted stones at Gesher Benot Ya‘aqov, Israel

PubMed Central

Goren-Inbar, Naama; Sharon, Gonen; Melamed, Yoel; Kislev, Mordechai

2002-01-01

The Acheulian site of Gesher Benot Ya‘aqov (Israel) has revealed a unique association of edible nuts with pitted hammers and anvils. Located in the Dead Sea rift, on the boundary between the Arabian and African plates, the site dates to the Early-Middle Pleistocene, oxygen isotope stage 19. In a series of strata, seven species of nuts, most of which can be cracked open only by a hard hammer, were uncovered. Five of the species are extant terrestrial nuts, and two are aquatic nuts now extinct in the Levant. In addition, the site yielded an assemblage of pitted hammers and anvils similar in pit morphology to those used by chimpanzees and contemporary hunter–gatherers. This is the first time, to our knowledge, that a site has offered both paleobotanical and lithic evidence of plant foods eaten by early hominins and technologies used for processing these foods. The evidence also sheds light on the structure of the community: ethnographic analogies suggest that mixedgender groups may have been active on the shores of paleoLake Hula. PMID:11854536

Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

PubMed Central

Ryner, L C; Takagaki, Y; Manley, J L

1989-01-01

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively. Images PMID:2566911

Strong metal-support interactions

NASA Technical Reports Server (NTRS)

Vannice, M. Albert

1987-01-01

It has been demonstrated that synergistic metal-support effects can occur which markedly enhance specific activity and alter selectivity in certain reactions. Because of the presence of such effects in certain reactions conducted under reducing conditions (that is, under H2 pressure), but not others, the creation of unique sites at the metal-support interface seems to be the best model at the present time to explain this behavior. The postulation of these sites, which are specific for a certain reactant such as CO, provides an effective explanation for the higher methanation rates that have been reported over some catalysts. The creation of these sites in the adlineation zone is facilitated by hydrogen spillover from the metal surface, and this same process can also enhance the reduction of many oxide supports. Although oxygen spillover is much less probable due to its higher heat of adsorption, it is much less well understood and the possibility of rate enhancements in CO oxidation caused by special interface sites cannot be discounted at the present time. Consequently, this seems to be an important area of future research.

Multiscalar approach to archaeological site formation at GaJj17, East Turkana, Kenya

NASA Astrophysics Data System (ADS)

Murray, B. M.; Ranhorn, K. L.; Colarossi, D.; Mavuso, S. S.; Dogandžić, T.; Ziegler, M. J.; Warren, S. L.; Braun, D. R.; Harris, J. W. K.

2017-12-01

Kenya's East Turkana region hosts a rich Plio­Pleistocene record of fossils, archaeological artifacts, and sedimentary features whose chronostratigraphic histories are often obscured by landscape changes from erosional events and tectonic activity. The Middle Stone Age (MSA) record of the Koobi Fora Formation (KF Fm.) has particularly been subjected to this complex depositional history, making it a sparse unit and, consequently, widely understudied. Stratigraphically located in between the maximum capping unconformity of the KF Fm.'s Chari tuff ( 1.39 Ma) and that of the Galana Boi Fm. ( 10 ka), the unit provides a unique window into understanding the Late Pleistocene of the region. The MSA surface scatters at archaeological site GaJj17 prompted further study into the site's age and depositional chronology. The GaJj17 ridge is locally distinguished by its cap of Late Pleistocene sands overlying strata containing tuffs likely of the Upper Burgi (2.0­-1.87 Ma) or KBS (1.87­-1.56 Ma) members. To investigate whether GaJj17's preservation is due to tectonic deformation, a broader scale examination of the structural geology was conducted through surveys and aerial imagery. Regions of deformation were identified and mapped to establish the geological history of the locality. Resultant observations and elevation data offer insight into regional faults at the root of prolonged structural alterations which have facilitated the unique preservation of MSA materials. Through a multiscalar approach it is possible to understand both the formation of GaJj17 and the underlying processes behind preservation and destruction in the changing landscape of the Turkana basin, enabling future identification of archaeological sites through proxies of elevation, regional stratigraphy, and fault mapping. This research was supported by IRES grants 1358178 and 1358200 from the U.S. National Science Foundation.

The dynamics of methane emissions in Alaskan peatlands at different trophic levels

NASA Astrophysics Data System (ADS)

Zhang, L.; Liu, X.; Langford, L.; Chanton, J.; Hines, M. E.

2016-12-01

One major uncertainty in estimating methane (CH4) emission from wetlands is extrapolating from highly heterogeneous and inadequately studied local sites to larger scales. The heterogeneity of peatlands comes from contrasting surface vegetation compositions within short distances that are usually associated with different nutrient sources and trophic status. Different microbial communities and metabolic pathways occur at different trophic levels. Stable isotope C ratios (δ13C) have been used as a robust tool to distinguish methanogenic pathways, but different sources of parent compounds (acetate and CO2) with unique δ13C signatures, and unresolved fractionation factors associated with different methanogens, add complexity. To better understand the relationships between trophic status, surface vegetation compositions and methanogenic pathways, 28 peatland sites were studied in Fairbanks and Anchorage, Alaska in the summer of 2015. These sites were ordinated using multiple factor analysis into 3 clusters based on pH, temp, CH4 and volatile fatty acids production rates, δ13C values, and surface vegetation composition. In the low-pH trophic cluster (pH 4.2), Sphagnum fuscum was the dominant species with specific sedges (Ledum decumbens), and primary fermentation rates was slow with no CH4 detected. In the intermediate trophic level (pH 5.3), in which Sphagnum magellanicum was largely present, both hydrogenotrophic (HM) and acetoclastic methanogenesis (AM) were very active. Syntrophy was present at certain sites, which may provide CO2 and acetate with unique δ13C for CH4 production. At the highest pH trophic cluster examined in this study (pH 5.8), Carex tenuiflora, Carex aquatilis, and Sphagnum Squarrosum dominated. CH4 production rates were higher than those in the intermediate cluster and the apparent fractionation factor a was lower.

Refining the Subseafloor Circulation Model of the Middle Valley Hydrothermal System Using Fluid Geochemistry

NASA Astrophysics Data System (ADS)

Inderbitzen, K. E.; Wheat, C. G.; Baker, P. A.; Fisher, A. T.

2014-12-01

Currently, fluid circulation patterns and the evolution of rock/fluid compositions as circulation occurs in subseafloor hydrothermal systems are poorly constrained. Sedimented spreading centers provide a unique opportunity to study subsurface flow because sediment acts as an insulating blanket that traps heat from the cooling magma body and limits: (a) potential flow paths for seawater to recharge the aquifer in permeable upper basaltic basement and (b) points of altered fluid egress. This also allows for a range of thermal and geochemical gradients to exist near the sediment-water interface. Models of fluid circulation patterns in this type of hydrologic setting have been generated (eg. Stein and Fisher, 2001); however fluid chemistry datasets have not previously been used to test the model's viability. We address this issue by integrating the existing circulation model with fluid compositional data collected from sediment pore waters and high temperature hydrothermal vents located in Middle Valley on the Juan de Fuca Ridge. Middle Valley hosts a variety of hydrologic regimes: including areas of fluid recharge (Site 855), active venting (Site 858/1036; Dead Dog vent field), recent venting (Site 856/1035; Bent Hill Massive Sulfide deposit) and a section of heavily sedimented basement located between recharge and discharge sites (Site 857). We will present new results based on thermal and geochemical data from the area of active venting (Sites 858 and 1036), that was collected during Ocean Drilling Program Legs 139 and 169 and a subsequent heat flow/gravity coring effort. These results illuminate fine scale controls on secondary recharge and fluid flow within the sediment section at Site 858/1036. The current status of high temperature vents in this area (based on observations made in July, 2014) will also be outlined.

Enhanced guide-RNA design and targeting analysis for precise CRISPR genome editing of single and consortia of industrially relevant and non-model organisms.

PubMed

Mendoza, Brian J; Trinh, Cong T

2018-01-01

Genetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation for synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions. To accommodate the use of CRISPR/Cas systems for genome editing across organisms, we have developed a novel method, named CRISPR Associated Software for Pathway Engineering and Research (CASPER), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases. Utilizing CASPER, we demonstrated a modest 2.4% and significant 30.2% improvement (F-test, P < 0.05) over the conventional methods for predicting on- and off-target activities, respectively. Further we used CASPER to develop novel applications in genome editing: multitargeting analysis (i.e. simultaneous multiple-site modification on a target genome with a sole guide-RNA requirement) and multispecies population analysis (i.e. guide-RNA design for genome editing across a consortium of organisms). Our analysis on a selection of industrially relevant organisms revealed a number of non-unique target sites associated with genes and transposable elements that can be used as potential sites for multitargeting. The analysis also identified shared and unshared targets that enable genome editing of single or multiple genomes in a consortium of interest. We envision CASPER as a useful platform to enhance the precise CRISPR genome editing for metabolic engineering and synthetic biology applications. https://github.com/TrinhLab/CASPER. ctrinh@utk.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Poroelastic Parameters of Peru Margin Sediments: Implications for Flow and Transport at Multiple Scales in the Marine Biosphere

NASA Astrophysics Data System (ADS)

Gettemy, G. L.; Cikoski, C.; Tobin, H. J.

2004-12-01

As part of a broader investigation of the deep marine subsurface environment, the first biosphere-focused drilling expedition, Leg 201, of the Ocean Drilling Program (ODP) occupied five unique sites in the Peru Margin (in a 1200 km2 region centered at 10 S, 80E). These sites represent the entire range of shallow biogeological conditions associated with this convergent margin:deep-water, mixed clay-pelagic sediments ocean-ward of the trench; slope-apron and prism toe sediments at the deformation front; and several distinct lithostratigraphic sequences on the continental shelf. Microbial enumeration and pore-water geochemistry results show that each particular site is both consistent and unique--consistent in terms of general biotic quantity and activity as predicted by energy flux and redox potential given the depositional environment and sedimentary record, but unique at key biogeological boundaries such as lithologic and/or physical property interfaces. This research addresses questions related to our understanding of how and why these boundaries form by looking at poroelastic and hydrologic parameters measured at multiple scales, from sub-millimeter to several centimeters. The issue of measurement scale, especially in regard to permeability and diffusivity characterization, is vital to interpreting observations of biologically-mediated diagenetic fronts (e.g., dolomitic lenses, depth- or time-varying barite fronts). These parameters are derived from (i) hydrologic and wave propagation experiments, (ii) SEM images, and (iii) shipboard split-core measurements, and structured in a modified Biot poroelasticity framework. This approach also allows quantification of the local heterogeneity of these parameters at the scale applicable to (and controlled by) microbial life; these results can then be used to formulate predictive models of the impact of biogeochemical processes. Ultimately, these models could then be used in interpretation of new remote-sensed data (e.g., from borehole tools, high-frequency backscatter devices), a fundamental challenge for all types of biospheric imaging everywhere.

The Copper Active Site of CBM33 Polysaccharide Oxygenases

PubMed Central

2013-01-01

The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

Role of hydrogen bonding in ligand interaction with the N-methyl-D-aspartate receptor ion channel

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

Leeson, P.D.; Carling, R.W.; James, K.

1990-05-01

Displacement of (3H)MK-801 (dizocilpine, 1) binding to rat brain membranes has been used to evaluate the affinities of novel dibenzocycloalkenimines related to 1 for the ion channel binding site (also known as the phencyclidine or PCP receptor) on the N-methyl-D-aspartate (NMDA) subtype of excitory amino acid receptor. In common with many other agents having actions in the central nervous system, these compounds contain a hydrophobic aromatic moiety and a basic nitrogen atom. The conformational rigidity of these ligands provides a unique opportunity to evaluate the importance of specific geometrical properties that influence active-site recognition, in particular the role of themore » nitrogen atom in hydrogen-bonding interactions. The relative affinities (IC50s) of hydrocarbon-substituted analogues of 1 and ring homologated cyclooctenimines illustrate the importance of size-limited hydrophobic binding of both aryl rings and of the quaternary C-5 methyl group. Analysis of the binding of a series of the 10 available structurally rigid dibenzoazabicyclo(x.y.z)alkanes, by using molecular modeling techniques, uncovered a highly significant correlation between affinity and a proposed ligand-active site hydrogen bonding vector (r = 0.950, p less than 0.001). These results are used to generate a pharmacophore of the MK-801 recognition site/PCP receptor, which accounts for the binding of all of the known ligands.« less

Understanding the ecological background of rice agriculture on the Ningshao Plain during the Neolithic Age: pollen evidence from a buried paddy field at the Tianluoshan cultural site

NASA Astrophysics Data System (ADS)

Li, Chunhai; Zheng, Yunfei; Yu, Shiyong; Li, Yongxiang; Shen, Huadong

2012-03-01

The progressive rise of atmospheric CH4 level since 5 ka has been hypothesized to result from human agricultural activities that turned forested lands, which would otherwise be a carbon sink, into paddy fields. Increasing numbers of Neolithic cultural sites unearthed in coastal eastern China, providing unique opportunities to test this hypothesis. Here, we present detailed pollen data from a buried paddy field at Tianluoshan cultural site on the Ningshao Plain, eastern China, to reconstruct the ecological conditions associated with the establishment of paddy fields. Stratigraphic data, radiocarbon ages, and pollen analyses show that vegetation underwent six phases of evolution and paddy fields were developed from 7000 to 4200 cal. yr BP. We found no evidence of slash-and-burn agriculture at the study site. Together with no presence of the irrigation system, our pollen data suggest the paddy fields at this site originated from wetlands. Hence, our findings do not support the hypothesis that anthropogenic-induced deforestation play ed a significant role in the rise of the atmospheric CH4 rise since the middle Holocene.

Development of the pseudothumb in frogs

PubMed Central

Tokita, Masayoshi; Iwai, Noriko

2010-01-01

Frogs have highly conserved hand and foot morphology, possessing four fingers and five toes. As an exception, two Japanese ranid frog species, the Otton frog Babina subaspera and the dagger frog Babina holsti, possess a unique thumb-like structure (the pseudothumb) in the forelimb, giving an appearance of a total of five fingers on the hand. To obtain insights into the developmental mechanisms that generate this novel character, we investigated the hand morphogenesis of the Otton frog. The unique morphological pattern of the pseudothumb was already established in juveniles. Surprisingly, the bud-like structure, which is similar to the area of inductive activity (e.g. feather buds in birds and the carapacial ridge in turtles), was detected over the site where the future prepollex develops in larvae. By contrast, this bud-like structure was not found in larvae of other ranid species. We discuss possible scenarios that would favour the evolution of this very unusual trait in frogs. PMID:20147308

Whooping crane stopover site use intensity within the Great Plains

USGS Publications Warehouse

Pearse, Aaron T.; Brandt, David A.; Harrell, Wade C.; Metzger, Kristine L.; Baasch, David M.; Hefley, Trevor J.

2015-09-23

Whooping cranes (Grus americana) of the Aransas-Wood Buffalo population migrate twice each year through the Great Plains in North America. Recovery activities for this endangered species include providing adequate places to stop and rest during migration, which are generally referred to as stopover sites. To assist in recovery efforts, initial estimates of stopover site use intensity are presented, which provide opportunity to identify areas across the migration range used more intensively by whooping cranes. We used location data acquired from 58 unique individuals fitted with platform transmitting terminals that collected global position system locations. Radio-tagged birds provided 2,158 stopover sites over 10 migrations and 5 years (2010–14). Using a grid-based approach, we identified 1,095 20-square-kilometer grid cells that contained stopover sites. We categorized occupied grid cells based on density of stopover sites and the amount of time cranes spent in the area. This assessment resulted in four categories of stopover site use: unoccupied, low intensity, core intensity, and extended-use core intensity. Although provisional, this evaluation of stopover site use intensity offers the U.S. Fish and Wildlife Service and partners a tool to identify landscapes that may be of greater conservation significance to migrating whooping cranes. Initially, the tool will be used by the U.S. Fish and Wildlife Service and other interested parties in evaluating the Great Plains Wind Energy Habitat Conservation Plan.

The dynamic disulphide relay of quiescin sulphydryl oxidase.

PubMed

Alon, Assaf; Grossman, Iris; Gat, Yair; Kodali, Vamsi K; DiMaio, Frank; Mehlman, Tevie; Haran, Gilad; Baker, David; Thorpe, Colin; Fass, Deborah

2012-08-16

Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 Å apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.

Evolution of nonspectral rhodopsin function at high altitudes.

PubMed

Castiglione, Gianni M; Hauser, Frances E; Liao, Brian S; Lujan, Nathan K; Van Nynatten, Alexander; Morrow, James M; Schott, Ryan K; Bhattacharyya, Nihar; Dungan, Sarah Z; Chang, Belinda S W

2017-07-11

High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Protein-level adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates-properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.

Identification of catalytic sites for oxygen reduction and oxygen evolution in N-doped graphene materials: Development of highly efficient metal-free bifunctional electrocatalyst

PubMed Central

Yang, Hong Bin; Miao, Jianwei; Hung, Sung-Fu; Chen, Jiazang; Tao, Hua Bing; Wang, Xizu; Zhang, Liping; Chen, Rong; Gao, Jiajian; Chen, Hao Ming; Dai, Liming; Liu, Bin

2016-01-01

Oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) are critical to renewable energy conversion and storage technologies. Heteroatom-doped carbon nanomaterials have been reported to be efficient metal-free electrocatalysts for ORR in fuel cells for energy conversion, as well as ORR and OER in metal-air batteries for energy storage. We reported that metal-free three-dimensional (3D) graphene nanoribbon networks (N-GRW) doped with nitrogen exhibited superb bifunctional electrocatalytic activities for both ORR and OER, with an excellent stability in alkaline electrolytes (for example, KOH). For the first time, it was experimentally demonstrated that the electron-donating quaternary N sites were responsible for ORR, whereas the electron-withdrawing pyridinic N moieties in N-GRW served as active sites for OER. The unique 3D nanoarchitecture provided a high density of the ORR and OER active sites and facilitated the electrolyte and electron transports. As a result, the as-prepared N-GRW holds great potential as a low-cost, highly efficient air cathode in rechargeable metal-air batteries. Rechargeable zinc-air batteries with the N-GRW air electrode in a two-electrode configuration exhibited an open-circuit voltage of 1.46 V, a specific capacity of 873 mAh g−1, and a peak power density of 65 mW cm−2, which could be continuously charged and discharged with an excellent cycling stability. Our work should open up new avenues for the development of various carbon-based metal-free bifunctional electrocatalysts of practical significance. PMID:27152333

CD and MCD of CytC3 and taurine dioxygenase: role of the facial triad in alpha-KG-dependent oxygenases.

PubMed

Neidig, Michael L; Brown, Christina D; Light, Kenneth M; Fujimori, Danica Galonić; Nolan, Elizabeth M; Price, John C; Barr, Eric W; Bollinger, J Martin; Krebs, Carsten; Walsh, Christopher T; Solomon, Edward I

2007-11-21

The alpha-ketoglutarate (alpha-KG)-dependent oxygenases are a large and diverse class of mononuclear non-heme iron enzymes that require FeII, alpha-KG, and dioxygen for catalysis with the alpha-KG cosubstrate supplying the additional reducing equivalents for oxygen activation. While these systems exhibit a diverse array of reactivities (i.e., hydroxylation, desaturation, ring closure, etc.), they all share a common structural motif at the FeII active site, termed the 2-His-1-carboxylate facial triad. Recently, a new subclass of alpha-KG-dependent oxygenases has been identified that exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl- ligand is coordinated in place of the carboxylate. An FeII methodology involving CD, MCD, and VTVH MCD spectroscopies was applied to CytC3 to elucidate the active-site structural effects of this perturbation of the coordination sphere. A significant decrease in the affinity of FeII for apo-CytC3 was observed, supporting the necessity of the facial triad for iron coordination to form the resting site. In addition, interesting differences observed in the FeII/alpha-KG complex relative to the cognate complex in other alpha-KG-dependent oxygenases indicate the presence of a distorted 6C site with a weak water ligand. Combined with parallel studies of taurine dioxygenase and past studies of clavaminate synthase, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the noncoordinating oxygen of the carboxylate and the coordinated water. These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.

Science@NASA: Direct to People!

NASA Technical Reports Server (NTRS)

Koczor, Ronald J.; Adams, Mitzi; Gallagher, Dennis; Whitaker, Ann (Technical Monitor)

2002-01-01

Science@NASA is a science communication effort sponsored by NASA's Marshall Space Flight Center. It is the result of a four year research project between Marshall, the University of Florida College of Journalism and Communications and the internet communications company, Bishop Web Works. The goals of Science@NASA are to inform, inspire, and involve people in the excitement of NASA science by bringing that science directly to them. We stress not only the reporting of the facts of a particular topic, but also the context and importance of the research. Science@NASA involves several levels of activity from academic communications research to production of content for 6 websites, in an integrated process involving all phases of production. A Science Communications Roundtable Process is in place that includes scientists, managers, writers, editors, and Web technical experts. The close connection between the scientists and the writers/editors assures a high level of scientific accuracy in the finished products. The websites each have unique characters and are aimed at different audience segments: 1. http://science.nasa.gov. (SNG) Carries stories featuring various aspects of NASA science activity. The site carries 2 or 3 new stories each week in written and audio formats for science-attentive adults. 2. http://liftoff.msfc.nasa.gov. Features stories from SNG that are recast for a high school level audience. J-Track and J-Pass applets for tracking satellites are our most popular product. 3. http://kids. msfc.nasa.gov. This is the Nursemaids site and is aimed at a middle school audience. The NASAKids Club is a new feature at the site. 4. http://www.thursdaysclassroom.com . This site features lesson plans and classroom activities for educators centered around one of the science stories carried on SNG. 5. http://www.spaceweather.com. This site gives the status of solar activity and its interactions with the Earth's ionosphere and magnetosphere.

Affinity alkylation of the active site of C/sub 21/ steroid side-chain cleavage cytochrome P-450 from neonatal porcine testis: a unique cysteine residue alkylated by 17-(bromoacetoxy)progesterone

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

Onoda, M.; Haniu, M.; Yanagibashi, K.

1987-01-27

The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C/sub 20/ and C/sub 21/ side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C/sub 17,20/-lyase reactions, which produce the corresponding C/sub 1//sup 9/ steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme against inactivation. The progesterone analogue is a competitivemore » inhibitor of the enzyme with K/sub i/ values of 8.4 ..mu..M and 7.8 ..mu..M for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-((/sup 14/C)bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabeled enzyme samples, from reactions of the /sup 14/C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also in tryptic digestion and peptide mapping.« less

Methane oxidation on Pd–Ceria: A DFT study of the mechanism over PdxCe1-xO2, Pd, and PdO

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

Mayernick, Adam D.; Janik, Michael J.

2011-02-14

Palladium/ceria exhibits unique catalytic activity for hydrocarbon oxidation; however, the chemical and structural properties of active sites on the palladium–ceria surface are difficult to characterize. Strong interactions between palladium and the ceria support stabilize oxidized Pdδ+ species, which may contribute to the significant activity of Pd/ceria for methane oxidation. We present a density functional theory (DFT + U) investigation into methane oxidation over Pd/ceria and quantify the activity of the Pd xCe 1-xO 2(1 1 1) mixed oxide surface in comparison with the PdO(1 0 0) and Pd(1 1 1) surfaces. The methane activation barrier is lowest over the Pdmore » xCe 1-xO 2(1 1 1) surface, even lower than over the Pd(1 1 1) surface or low coordinated stepped or kinked Pd sites. Subsequent reaction steps in complete oxidation, including product desorption and vacancy refilling, are considered to substantiate that methane activation remains the rate-limiting step despite the low barrier over Pd xCe 1-xO 2(1 1 1). The low barrier over the Pd xCe 1-xO 2(1 1 1) surface demonstrates that mixed ceria-noble metal oxides offer the potential for improved hydrocarbon oxidation performance with respect to dispersed noble metal particles on ceria.« less

Water oxidation by Ruthenium complexes incorporating multifunctional biipyridyl diphosphonate ligands

DOE PAGES

Xie, Yan; Shaffer, David W.; Lewandowska-Andralojc, Anna; ...

2016-05-11

Here, we describe herein the synthesis and characterization of ruthenium complexes with multifunctional bipyridyl diphosphonate ligands as well as initial water oxidation studies. In these complexes, the phosphonate groups provide redox-potential leveling through charge compensation and σ donation to allow facile access to high oxidation states. These complexes display unique pH-dependent electrochemistry associated with deprotonation of the phosphonic acid groups. The position of these groups allows them to shuttle protons in and out of the catalytic site and reduce activation barriers. A mechanism for water oxidation by these catalysts is proposed on the basis of experimental results and DFT calculations.more » The unprecedented attack of water at a neutral six-coordinate [Ru IV] center to yield an anionic seven-coordinate [Ru IV–OH] – intermediate is one of the key steps of a single-site mechanism in which all species are anionic or neutral. These complexes are among the fastest single-site catalysts reported to date.« less

American black bear denning behavior: Observations and applications using remote photography

USGS Publications Warehouse

Bridges, A.S.; Fox, J.A.; Olfenbuttel, C.; Vaughan, M.B.

2004-01-01

Researchers examining American black bear (Ursus americanus) denning behavior have relied primarily on den-site visitation and radiotelemetry to gather data. Repeated den-site visits are time-intensive and may disturb denning bears, possibly causing den abandonment, whereas radiotelemetry is sufficient only to provide gross data on den emergence. We used remote cameras to examine black bear denning behavior in the Allegheny Mountains of western Virginia during March-May 2003. We deployed cameras at 10 den sites and used 137 pictures of black bears. Adult female black bears exhibited greater extra-den activity than we expected prior to final den emergence, which occurred between April 12 and May 6, 2003. Our technique provided more accurate den-emergence estimation than previously published methodologies. Additionally, we observed seldom-documented behaviors associated with den exits and estimated cub age at den emergence. Remote cameras can provide unique insights into denning ecology, and we describe their potential application to reproductive, survival, and behavioral research.

Bone Genes in the Kidney of Stone Formers

NASA Astrophysics Data System (ADS)

Evan, Andrew P.; Bledsoe, Sharon B.

2008-09-01

Intraoperative papillary biopsies from kidneys of idiopathic-calcium oxalate stone formers (ICSF) have revealed a distinct pattern of mineral deposition in the interstitium of the renal papilla. The earliest sites of these deposits, termed Randall's plaque, are found in the basement membrane of thin loops of Henle and appear to spread into the surrounding interstitium down to the papillary epithelium. Recent studies show kidney stones of ICSF patients grow attached to the renal papilla and at sites of Randall's plaque. Together these observations suggest that plaque formation may be the critical step in stone formation. In order to control plaque formation and thereby reduce future kidney stone development, the mechanism of plaque deposition must be understood. Because the renal papilla has unique anatomical features similar to bone and the fact that the interstitial deposits of ICSF patients are formed of biological apatite, this paper tests the hypothesis that sites of interstitial plaque form as a result of cell-mediated osteoblast-like activity.

Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

PubMed Central

Sundaram, Vasavi; Choudhary, Mayank N. K.; Pehrsson, Erica; Xing, Xiaoyun; Fiore, Christopher; Pandey, Manishi; Maricque, Brett; Udawatta, Methma; Ngo, Duc; Chen, Yujie; Paguntalan, Asia; Ray, Tammy; Hughes, Ava; Cohen, Barak A.; Wang, Ting

2017-01-01

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. PMID:28348391

Binding and Inactivation Mechanism of a Humanized Fatty Acid Amide Hydrolase by [alpha]-Ketoheterocycle Inhibitors Revealed from Cocrystal Structures

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

Mileni, Mauro; Garfunkle, Joie; DeMartino, Jessica K.

The cocrystal X-ray structures of two isomeric {alpha}-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional 'in-action' depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolicmore » port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.« less

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides.

PubMed

Bowen, Anthony; Wear, Maggie P; Cordero, Radames J B; Oscarson, Stefan; Casadevall, Arturo

2017-01-13

Studies in the 1980s first showed that some natural antibodies were "catalytic" and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides*

PubMed Central

Wear, Maggie P.; Cordero, Radames J. B.; Oscarson, Stefan

2017-01-01

Studies in the 1980s first showed that some natural antibodies were “catalytic” and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. PMID:27872188

Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase.

PubMed

Paradisi, Francesca; Dean, Jonathan L E; Geoghegan, Kieran F; Engel, Paul C

2005-03-08

A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis.

Development of a biomarker for Geobacter activity and strain composition: Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

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

Wilkins, M.J.; Callister, S.J.; Miletto, M.

2010-02-15

Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situmore » biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.« less

Development of a biomarker for Geobacter activity and strain composition; Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).

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

Wilkins, Michael J.; Callister, Stephen J.; Miletto, Marzia

2011-01-01

Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the U.S. Department of Energy’s Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situmore » biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.« less

The rules of engagement: comparing two social protest movements on YouTube.

PubMed

Vraga, Emily K; Bode, Leticia; Wells, Chris; Driscoll, Kevin; Thorson, Kjerstin

2014-03-01

Social media Web sites such as YouTube offer activists unique opportunities to reach out to new audiences through a variety of diverse appeals. Yet the rules of engagement on social media should depend on the structures, goals, and characteristics of the movements engaging in this outreach. To explore how differences in social movements translate into online activism, we employ a paired case study approach, comparing YouTube artifacts for two political mobilizations: the Occupy Movement and California's Proposition 8 ballot initiative concerning same sex marriage. Across movements, we examine the popularity of videos and their characteristics, and whether the type of video consistently predicts video engagement. We find that "social media activism" is not a unitary phenomenon; the two mobilizations produced unique YouTube ecologies. Occupy Wall Street videos tended on average to produce less engagement and focused on filmed live events and amateur content. Meanwhile, Proposition 8 videos usually produced more engagement and bridged more diverse formats: from professionalized and scripted content to live event footage and unscripted monologues to the camera. Therefore, our study suggests that social activism in online spaces such as YouTube is not easily defined, but is adapted to suit movement needs-which makes social media a popular and flexible venue for activism but also highlights the challenges for scholars studying such venues.

Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

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

Jin, R.; Sikorra, S.; Stegmann, C.M.

2009-06-01

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognitionmore » and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.« less

Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

PubMed

Yang, Xinmei; Matsui, Takashi; Kodama, Takeshi; Mori, Takahiro; Zhou, Xiaoxi; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

2016-03-01

In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+β barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-β-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future. Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively. © 2016 Federation of European Biochemical Societies.

A biosensor generated via high throughput screening quantifies cell edge Src dynamics

PubMed Central

Gulyani, Akash; Vitriol, Eric; Allen, Richard; Wu, Jianrong; Gremyachinskiy, Dmitriy; Lewis, Steven; Dewar, Brian; Graves, Lee M.; Kay, Brian K.; Kuhlman, Brian; Elston, Tim; Hahn, Klaus M.

2011-01-01

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge. PMID:21666688

High-resolution non-contact measurement of the electrical activity of plants in situ using optical recording

PubMed Central

Zhao, Dong-Jie; Chen, Yang; Wang, Zi-Yang; Xue, Lin; Mao, Tong-Lin; Liu, Yi-Min; Wang, Zhong-Yi; Huang, Lan

2015-01-01

The limitations of conventional extracellular recording and intracellular recording make high-resolution multisite recording of plant bioelectrical activity in situ challenging. By combining a cooled charge-coupled device camera with a voltage-sensitive dye, we recorded the action potentials in the stem of Helianthus annuus and variation potentials at multiple sites simultaneously with high spatial resolution. The method of signal processing using coherence analysis was used to determine the synchronization of the selected signals. Our results provide direct visualization of the phloem, which is the distribution region of the electrical activities in the stem and leaf of H. annuus, and verify that the phloem is the main action potential transmission route in the stems of higher plants. Finally, the method of optical recording offers a unique opportunity to map the dynamic bioelectrical activity and provides an insight into the mechanisms of long-distance electrical signal transmission in higher plants. PMID:26333536

Chemoproteomics reveals baicalin activates hepatic CPT1 to ameliorate diet-induced obesity and hepatic steatosis.

PubMed

Dai, Jianye; Liang, Kai; Zhao, Shan; Jia, Wentong; Liu, Yuan; Wu, Hongkun; Lv, Jia; Cao, Chen; Chen, Tao; Zhuang, Shentian; Hou, Xiaomeng; Zhou, Shijie; Zhang, Xiannian; Chen, Xiao-Wei; Huang, Yanyi; Xiao, Rui-Ping; Wang, Yan-Ling; Luo, Tuoping; Xiao, Junyu; Wang, Chu

2018-06-11

Obesity and related metabolic diseases are becoming worldwide epidemics that lead to increased death rates and heavy health care costs. Effective treatment options have not been found yet. Here, based on the observation that baicalin, a flavonoid from the herbal medicine Scutellaria baicalensis , has unique antisteatosis activity, we performed quantitative chemoproteomic profiling and identified carnitine palmitoyltransferase 1 (CPT1), the controlling enzyme for fatty acid oxidation, as the key target of baicalin. The flavonoid directly activated hepatic CPT1 with isoform selectivity to accelerate the lipid influx into mitochondria for oxidation. Chronic treatment of baicalin ameliorated diet-induced obesity (DIO) and hepatic steatosis and led to systemic improvement of other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 completely abolished the beneficial effect of the flavonoid. Our discovery of baicalin as an allosteric CPT1 activator opens new opportunities for pharmacological treatment of DIO and associated sequelae.

Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease.

PubMed

Kagawa, T F; Cooney, J C; Baker, H M; McSweeney, S; Liu, M; Gubba, S; Musser, J M; Baker, E N

2000-02-29

Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes.

Performance Art at the Campusphere: Pedagogical Experiments On-Site

ERIC Educational Resources Information Center

Ben-Shaul, Daphna

2018-01-01

Following a unique practice and research laboratory entitled "Performance: Site/Self" that took place in 2013-2015, this article discusses the implementation of performance art at an academic site--the Tel Aviv University campus. This pedagogical and artistic initiative, characterised by the transgressive pedagogy of performance art…

Detection of the CLOCK/BMAL1 heterodimer using a nucleic acid probe with cycling probe technology.

PubMed

Nakagawa, Kazuhiro; Yamamoto, Takuro; Yasuda, Akio

2010-09-15

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms. 2010 Elsevier Inc. All rights reserved.

Face to (face)book: the two faces of social behavior?

PubMed

Ivcevic, Zorana; Ambady, Nalini

2013-06-01

Social networking sites such as Facebook represent a unique and dynamic social environment. This study addresses three theoretical issues in personality psychology in the context of online social networking sites: (a) the temporal consistency of Facebook activity, (b) people's awareness of their online behavior, and (c) comparison of social behavior on Facebook with self- and informant-reported behavior in real life. Facebook Wall pages of 99 college students (mean age = 19.72) were downloaded six times during 3 weeks and coded for quantity and quality of activity. Everyday social interactions were assessed by self- and friend report. Facebook activity showed significant consistency across time, and people demonstrated awareness of their online behavior. There was significant similarity between everyday traits and interactions and Facebook behavior (e.g., more posts by friends are related to Agreeableness). Some differences between online and everyday interactions warrant further research (e.g., individuals with more positive offline relationships are less likely to engage in back-and-forth conversations on Facebook). The results indicate substantial similarity between online and offline social behavior and identify avenues for future research on the possible use of Facebook to compensate for difficulty in everyday interactions. © 2012 Wiley Periodicals, Inc.

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling.

PubMed

Ji, Qinqin; Ding, Yiyuan; Salomon, Arthur R

2015-01-01

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

SRC Homology 2 Domain-containing Leukocyte Phosphoprotein of 76 kDa (SLP-76) N-terminal Tyrosine Residues Regulate a Dynamic Signaling Equilibrium Involving Feedback of Proximal T-cell Receptor (TCR) Signaling*

PubMed Central

Ji, Qinqin; Ding, Yiyuan; Salomon, Arthur R.

2015-01-01

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor–mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr112, Tyr128, and Tyr145, in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr192 of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr440 of Fyn, Tyr702 of PLCγ1, Tyr204, Tyr397, and Tyr69 of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues. PMID:25316710

Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

PubMed Central

Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

2011-01-01

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

HDAC inhibitors TSA and sodium butyrate enhanced the human IL-5 expression by altering histone acetylation status at its promoter region.

PubMed

Han, Songyan; Lu, Jun; Zhang, Yu; Cheng, Cao; Li, Lin; Han, Liping; Huang, Baiqu

2007-02-15

The expression of IL-5 correlated tightly with the maturation and differentiation of eosinophils, and is considered as a cytokine responsible for allergic inflammation. We report here that inhibition of HDAC activity by Trichostatin A (TSA) and sodium butyrate (NaBu), the two specific HDAC inhibitors, resulted in the elevation of both endogenous and exogenous activity of IL-5 promoter. We demonstrated that both the mRNA expression and protein production of IL-5 were stimulated by TSA and NaBu treatments. ChIP assays showed that treatments of TSA and NaBu caused hyperacetylation of histones H3 and H4 on IL-5 promoter in Jurkat cells, which consequently promoted the exogenous luciferase activity driven by this promoter. Moreover, site-directed mutagenesis studies showed that the binding sites for transcription factors NFAT, GATA3 and YY1 on IL-5 promoter were critical for the effects of TSA and NaBu, suggesting that the transcriptional activation of IL-5 gene by these inhibitors was achieved by affecting HDAC function on IL-5 promoter via transcription factors. These data will contribute to elucidating the unique mechanism of IL-5 transcriptional control and to the therapy of allergic disorders related to IL-5.

Boronic acid-containing CXCR1/2 antagonists: Optimization of metabolic stability, in vivo evaluation, and a proposed receptor binding model.

PubMed

Maeda, Dean Y; Peck, Angela M; Schuler, Aaron D; Quinn, Mark T; Kirpotina, Liliya N; Wicomb, Winston N; Auten, Richard L; Gundla, Rambabu; Zebala, John A

2015-06-01

Blockade of undesired neutrophil migration to sites of inflammation remains an area of substantial pharmaceutical interest. To effect this blockade, a validated therapeutic target is antagonism of the chemokine receptor CXCR2. Herein we report the discovery of 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide 6, an antagonist with activity at both CXCR1 and CXCR2 receptors (IC50 values 31 and 21 nM, respectively). Compound 6 exhibited potent inhibition of neutrophil influx in a rat model of pulmonary inflammation, and is hypothesized to interact with a unique intracellular binding site on CXCR2. Compound 6 (SX-576) is undergoing further investigation as a potential therapy for pulmonary inflammation. Copyright © 2015 Elsevier Ltd. All rights reserved.

The lost micro-deserts of the Patuxent River using landscape history, insect and plant specimens, and field work to detect and define a unique community

USGS Publications Warehouse

Droege, S.; Davis, C.A.; Steiner, W.E.; =Mawdsley, J.

2009-01-01

Historical and recent records of both plants and insects are synthesized for uplands along the eastern edge of Maryland?s Patuxent River from the edge of the Piedmont south to Jug Bay. This strip is characterized by deep sandy soils found in the Evesboro and Galestown sandy loams soil series. Within this narrow strip there exists a unique flora and fauna adapted to open dry sandy soils and occurring in small remnant patches associated with old sand mining operations and scattered protected areas. We illustrate the uniqueness of these sites using four groups, vascular plants, tenebrionid beetles (Coleoptera: Tenebrionidae), tiger beetles (Coleoptera: Cicindelidae), and bees (Hymenoptera: Apoidea: Anthophila). Within each of these groups, rare species were detected whose populations were locally restricted to this soil type and whose nearest known populations were often hundreds of kilometers away. In addition to documenting the direct conservation importance of these small sandy openings along the Patuxent, we contrast the lack of any indication from vertebrate inventories that this region is unique. The combination of plant and insect inventories appears to be a better means of clarifying a site?s importance than does any survey of a single taxonomic group.

Hydrologic Source Term Processes and Models for the Clearwater and Wineskin Tests, Rainier Mesa, Nevada National Security Site

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

Carle, Steven F.

2011-05-04

This report describes the development, processes, and results of a hydrologic source term (HST) model for the CLEARWATER (U12q) and WINESKIN (U12r) tests located on Rainier Mesa, Nevada National Security Site, Nevada (Figure 1.1). Of the 61 underground tests (involving 62 unique detonations) conducted on Rainier Mesa (Area 12) between 1957 and 1992 (USDOE, 2015), the CLEARWATER and WINESKIN tests present many unique features that warrant a separate HST modeling effort from other Rainier Mesa tests.

16 CFR 1031.18 - Method of review and comment.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Commission staff is involved shall have a unique Web link on the Commission Web site with relevant... forwarded to appropriate staff for consideration and/or response. (c) On the voluntary standards Web site...

16 CFR 1031.18 - Method of review and comment.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Commission staff is involved shall have a unique Web link on the Commission Web site with relevant... forwarded to appropriate staff for consideration and/or response. (c) On the voluntary standards Web site...

16 CFR 1031.18 - Method of review and comment.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Commission staff is involved shall have a unique Web link on the Commission Web site with relevant... forwarded to appropriate staff for consideration and/or response. (c) On the voluntary standards Web site...

Proteomic content of circulating exosomes in dairy cows with or without uterine infection.

PubMed

Almughlliq, Fatema B; Koh, Yong Q; Peiris, Hassendrini N; Vaswani, Kanchan; McDougall, Scott; Graham, Elizabeth M; Burke, Chris R; Arachchige, Buddhika J; Reed, Sarah; Mitchell, Murray D

2018-07-01

In the past few decades, there has been a global decrease in dairy cow reproductive performance. An activated inflammatory system, due to uterine infection, has been associated with decreased cow fertility and as such, there is a need to detect uterine disease earlier. Early detection could be achieved by identifying biomarkers for uterine disease. Exosomes are small nanovesicles known to package and deliver protein, mRNA, and miRNAs to near and distant sites. Therefore, the content of circulating exosomes may have the potential to carry biomarkers for earlier diagnosis of disease. We hypothesized that circulating exosomes from cows with and without uterine infection may contain information representative of endometrial health or disease. We compared the proteomic content of circulating exosomes derived from plasma of dairy cows with (n = 10) or without (n = 10) induced uterine infection, using high-performance liquid chromatography tandem mass spectrometry (HPLC MS/MS). Our results demonstrate that there were a total of 103 bovine and 9 Trueperella pyogenes proteins found in plasma exosomes derived from infected cows (infected exosomes), and 90 bovine and 5 T. pyogenes proteins found in exosomes derived from plasma of non-infected cows (non-infected exosomes). 71 bovine proteins were found to be unique to the infected exosomes while only 4 bovine proteins were found to be unique to the non-infected exosomes. 8 unique T. pyogenes proteins were identified in infected exosomes and 4 were found to be unique to the non-infected exosomes. Pathway analysis showed that infected exosomes had more proteins involved in structural molecule activity and immune system processes than non-infected exosomal protein. Additionally, proteins from infected exosomes were involved in unique pathways: angiogenesis and integrin signaling pathway. Our data provide preliminary evidence of a potential role for exosomes in the early diagnosis of uterine infection in dairy cows. Copyright © 2018 Elsevier Inc. All rights reserved.

Solvothermal synthesis of stable nanoporous polymeric bases-crystalline TiO2 nanocomposites: visible light active and efficient photocatalysts for water treatment

NASA Astrophysics Data System (ADS)

Liu, Fujian; Kong, Weiping; Wang, Liang; Noshadi, Iman; Zhang, Zhonghua; Qi, Chenze

2015-02-01

Visible light active and stable nanoporous polymeric base-crystalline TiO2 nanocomposites were solvothermally synthesized from in situ copolymerization of divinylbenzene (DVB) with 1-vinylimidazolate (VI) or 4-vinylpyridine (Py) in the presence of tetrabutyl titanate without the use of any other additives (PDVB-VI-TiO2-x, PDVB-Py-TiO2-x, where x stands for the molar ratio of TiO2 to VI or Py), which showed excellent activity with respect to catalyzing the degradation of organic pollutants of p-nitrophenol (PNP) and rhodamine-B (RhB). TEM and SEM images show that PDVB-VI-TiO2-x and PDVB-Py-TiO2-x have abundant nanopores, and TiO2 nanocrystals with a high degree of crystallinity were homogeneously embedded in the PDVB-VI-TiO2-x and PDVB-Py-TiO2-x, forming a stable ‘brick-and-mortar’ nanostructure. PDVB-VI and PDVB-Py supports act as the glue linking TiO2 nanocrystals to form nanopores and constraining the agglomeration of TiO2 nanocrystals. XPS spectra show evidence of unique interactions between TiO2 and basic sites in these samples. UV diffuse reflectance shows that PDVB-VI-TiO2-x and PDVB-Py-TiO2-x exhibit a unique response to visible light. Catalytic tests show that the PDVB-VI-TiO2-x and PDVB-Py-TiO2-x were active in catalyzing the degradation of PNP and RhB organic pollutants under visible light irradiation. The enhanced activities of the PDVB-VI-TiO2-x and PDVB-Py-TiO2-x were ascribed to synergistic effects between abundant nanopores and the unique optical adsorption of visible light in the samples.

Solvothermal synthesis of stable nanoporous polymeric bases-crystalline TiO2 nanocomposites: visible light active and efficient photocatalysts for water treatment.

PubMed

Liu, Fujian; Kong, Weiping; Wang, Liang; Noshadi, Iman; Zhang, Zhonghua; Qi, Chenze

2015-02-27

Visible light active and stable nanoporous polymeric base-crystalline TiO2 nanocomposites were solvothermally synthesized from in situ copolymerization of divinylbenzene (DVB) with 1-vinylimidazolate (VI) or 4-vinylpyridine (Py) in the presence of tetrabutyl titanate without the use of any other additives (PDVB-VI-TiO2-x, PDVB-Py-TiO2-x, where x stands for the molar ratio of TiO2 to VI or Py), which showed excellent activity with respect to catalyzing the degradation of organic pollutants of p-nitrophenol (PNP) and rhodamine-B (RhB). TEM and SEM images show that PDVB-VI-TiO2-x and PDVB-Py-TiO2-x have abundant nanopores, and TiO2 nanocrystals with a high degree of crystallinity were homogeneously embedded in the PDVB-VI-TiO2-x and PDVB-Py-TiO2-x, forming a stable 'brick-and-mortar' nanostructure. PDVB-VI and PDVB-Py supports act as the glue linking TiO2 nanocrystals to form nanopores and constraining the agglomeration of TiO2 nanocrystals. XPS spectra show evidence of unique interactions between TiO2 and basic sites in these samples. UV diffuse reflectance shows that PDVB-VI-TiO2-x and PDVB-Py-TiO2-x exhibit a unique response to visible light. Catalytic tests show that the PDVB-VI-TiO2-x and PDVB-Py-TiO2-x were active in catalyzing the degradation of PNP and RhB organic pollutants under visible light irradiation. The enhanced activities of the PDVB-VI-TiO2-x and PDVB-Py-TiO2-x were ascribed to synergistic effects between abundant nanopores and the unique optical adsorption of visible light in the samples.

The Contributions of Near Work and Outdoor Activity to the Correlation Between Siblings in the Collaborative Longitudinal Evaluation of Ethnicity and Refractive Error (CLEERE) Study

PubMed Central

Jones-Jordan, Lisa A.; Sinnott, Loraine T.; Graham, Nicholas D.; Cotter, Susan A.; Kleinstein, Robert N.; Manny, Ruth E.; Mutti, Donald O.; Twelker, J. Daniel; Zadnik, Karla

2014-01-01

Purpose. We determined the correlation between sibling refractive errors adjusted for shared and unique environmental factors using data from the Collaborative Longitudinal Evaluation of Ethnicity and Refractive Error (CLEERE) Study. Methods. Refractive error from subjects' last study visits was used to estimate the intraclass correlation coefficient (ICC) between siblings. The correlation models used environmental factors (diopter-hours and outdoor/sports activity) assessed annually from parents by survey to adjust for shared and unique environmental exposures when estimating the heritability of refractive error (2*ICC). Results. Data from 700 families contributed to the between-sibling correlation for spherical equivalent refractive error. The mean age of the children at the last visit was 13.3 ± 0.90 years. Siblings engaged in similar amounts of near and outdoor activities (correlations ranged from 0.40–0.76). The ICC for spherical equivalent, controlling for age, sex, ethnicity, and site was 0.367 (95% confidence interval [CI] = 0.304, 0.420), with an estimated heritability of no more than 0.733. After controlling for these variables, and near and outdoor/sports activities, the resulting ICC was 0.364 (95% CI = 0.304, 0.420; estimated heritability no more than 0.728, 95% CI = 0.608, 0.850). The ICCs did not differ significantly between male–female and single sex pairs. Conclusions. Adjusting for shared family and unique, child-specific environmental factors only reduced the estimate of refractive error correlation between siblings by 0.5%. Consistent with a lack of association between myopia progression and either near work or outdoor/sports activity, substantial common environmental exposures had little effect on this correlation. Genetic effects appear to have the major role in determining the similarity of refractive error between siblings. PMID:25205866

Heterogeneous electrochemical CO2 reduction using nonmetallic carbon-based catalysts: current status and future challenges

NASA Astrophysics Data System (ADS)

Ma, Tao; Fan, Qun; Tao, Hengcong; Han, Zishan; Jia, Mingwen; Gao, Yunnan; Ma, Wangjing; Sun, Zhenyu

2017-11-01

Electrochemical CO2 reduction (ECR) offers an important pathway for renewable energy storage and fuels production. It still remains a challenge in designing highly selective, energy-efficient, robust, and cost-effective electrocatalysts to facilitate this kinetically slow process. Metal-free carbon-based materials have features of low cost, good electrical conductivity, renewability, diverse structure, and tunability in surface chemistry. In particular, surface functionalization of carbon materials, for example by doping with heteroatoms, enables access to unique active site architectures for CO2 adsorption and activation, leading to interesting catalytic performances in ECR. We aim to provide a comprehensive review of this category of metal-free catalysts for ECR, providing discussions and/or comparisons among different nonmetallic catalysts, and also possible origin of catalytic activity. Fundamentals and some future challenges are also described.

A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569.

PubMed

Fay, Jonathan F; Farrens, David L

2012-09-28

Allosteric ligands that modulate how G protein-coupled receptors respond to traditional orthosteric drugs are an exciting and rapidly expanding field of pharmacology. An allosteric ligand for the cannabinoid receptor CB1, Org 27569, exhibits an intriguing effect; it increases agonist binding, yet blocks agonist-induced CB1 signaling. Here we explored the mechanism behind this behavior, using a site-directed fluorescence labeling approach. Our results show that Org 27569 blocks conformational changes in CB1 that accompany G protein binding and/or activation, and thus inhibit formation of a fully active CB1 structure. The underlying mechanism behind this behavior is that simultaneous binding of Org 27569 produces a unique agonist-bound conformation, one that may resemble an intermediate structure formed on the pathway to full receptor activation.

Trans-activation of the Tetrahymena group I intron ribozyme via a non-native RNA-RNA interaction.

PubMed Central

Ikawa, Y; Shiraishi, H; Inoue, T

1999-01-01

The peripheral P2.1 domain of the Tetrahymena group I intron ribozyme has been shown to be non-essential for splicing. We found, however, that separately prepared P2.1 RNA efficiently accelerates the 3' splice-site-specific hydrolysis reaction of a mutant ribozyme lacking both P2.1 and its upstream region in trans. We report here the unusual properties of this trans-activation. Compensatory mutational analysis revealed that non-native long-range base-pairings between the loop region of P2.1 RNA and L5c region of the mutant ribozyme are needed for the activation in spite of the fact that P2.1 forms base-pairings with P9.1 in the Tetrahymena ribozyme. The trans -activation depends on the non-native RNA-RNA interaction together with the higher order structure of P2.1 RNA. This activation is unique among the known trans-activations that utilize native tertiary interactions or RNA chaperons. PMID:10075996

Session 35 - Panel: Remaining US Disposition Issues for Orphan or Small Volume Low Level and Low Level Mixed Waste Streams

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

Blauvelt, Richard; Small, Ken; Gelles, Christine

2006-07-01

Faced with closure schedules as a driving force, significant progress has been made during the last 2 years on the disposition of DOE mixed waste streams thought previously to be problematic. Generators, the Department of Energy and commercial vendors have combined to develop unique disposition paths for former orphan streams. Recent successes and remaining issues will be discussed. The session will also provide an opportunity for Federal agencies to share lessons learned on low- level and mixed low-level waste challenges and identify opportunities for future collaboration. This panel discussion was organized by PAC member Dick Blauvelt, Navarro Research and Engineeringmore » Inc who served as co-chair along with Dave Eaton from INL. In addition, George Antonucci, Duratek Barnwell and Rich Conley, AFSC were invited members of the audience, prepared to contribute the Barnwell and DOD perspective to the issues as needed. Mr. Small provide information regarding the five year 20K M3 window of opportunity at the Nevada Test Site for DOE contractors to dispose of mixed waste that cannot be received at the Energy Solutions (Envirocare) site in Utah because of activity levels. He provided a summary of the waste acceptance criteria and the process sites must follow to be certified to ship. When the volume limit or time limit is met, the site will undergo a RCRA closure. Ms. Gelles summarized the status of the orphan issues, commercial options and the impact of the EM reorganization on her program. She also announced that there would be a follow-on meeting in 2006 to the very successful St. Louis meeting of last year. It will probably take place in Chicago in July. Details to be announced. Mr. McKenney discussed progress made at the Hanford Reservation regarding disposal of their mixed waste inventory. The news is good for the Hanford site but not good for the rest of the DOE complex since shipment for out of state of both low level and low level mixed waste will continue to be prohibited until the completion of a new NEPA study. This is anticipated to take several years. Bill Franz from Portsmouth and Dave Eaton representing the INL provided the audience with information regarding some of the problematic mixed waste streams at their respective sites. Portsmouth has some unique radiological issues with isotopes such as Tc-99 while the INL is trying to deal with mixed waste in the 10-100 nCi/g range. Kaylin Loveland spoke of the new,Energy Solutions organization and provided information on mixed waste treatment capabilities at the Clive site. Mike Lauer described the licensing activities at the WCS site in Texas where they are trying to eventually have disposal capabilities for Class A, B and C mixed waste from both DOE and the commercial sector. The audience included about 75 WM'06 attendees who asked some excellent questions and provided an active and informative exchange of information on the topic. (authors)« less

Structural and Functional Analysis of DDX41: a bispecific immune receptor for DNA and cyclic dinucleotide

PubMed Central

Omura, Hiroki; Oikawa, Daisuke; Nakane, Takanori; Kato, Megumi; Ishii, Ryohei; Ishitani, Ryuichiro; Tokunaga, Fuminori; Nureki, Osamu

2016-01-01

In the innate immune system, pattern recognition receptors (PRRs) specifically recognize ligands derived from bacteria or viruses, to trigger the responsible downstream pathways. DEAD box protein 41 (DDX41) is an intracellular PRR that triggers the downstream pathway involving the adapter STING, the kinase TBK1, and the transcription factor IRF3, to activate the type I interferon response. DDX41 is unique in that it recognizes two different ligands; i.e., double-stranded DNA (dsDNA) and cyclic dinucleotides (CDN), via its DEAD domain. However, the structural basis for the ligand recognition by the DDX41 DEAD domain has remained elusive. Here, we report two crystal structures of the DDX41 DEAD domain in apo forms, at 1.5 and 2.2 Å resolutions. A comparison of the two crystal structures revealed the flexibility in the ATP binding site, suggesting its formation upon ATP binding. Structure-guided functional analyses in vitro and in vivo demonstrated the overlapped binding surface for dsDNA and CDN, which is distinct from the ATP-binding site. We propose that the structural rearrangement of the ATP binding site is crucial for the release of ADP, enabling the fast turnover of DDX41 for the dsDNA/CDN-induced STING activation pathway. PMID:27721487

Structure of D-tagatose 3-epimerase-like protein from Methanocaldococcus jannaschii.

PubMed

Uechi, Keiko; Takata, Goro; Yoneda, Kazunari; Ohshima, Toshihisa; Sakuraba, Haruhiko

2014-07-01

The crystal structure of a D-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the D-tagatose 3-epimerase from Pseudomonas cichorii and the D-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. However, the situation at the subunit-subunit interface differed substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV.

Structure of d-tagatose 3-epimerase-like protein from Methanocaldococcus jannaschii

PubMed Central

Uechi, Keiko; Takata, Goro; Yoneda, Kazunari; Ohshima, Toshihisa; Sakuraba, Haruhiko

2014-01-01

The crystal structure of a d-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the d-tagatose 3-epimerase from Pseudomonas cichorii and the d-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. However, the situation at the subunit–subunit interface differed substantially from that in d-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of d-tagatose 3-epimerase family enzymes and endonuclease IV. PMID:25005083

High-Resolution Mapping of Changes in Histone-DNA Contacts of Nucleosomes Remodeled by ISW2

PubMed Central

Kassabov, Stefan R.; Henry, Nathalia M.; Zofall, Martin; Tsukiyama, Toshio; Bartholomew, Blaine

2002-01-01

The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA. The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site-specific photochemical cross-linking approach. The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed. Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template. In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting. ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA. PMID:12370299

Metastable Atom-Activated Dissociation Mass Spectrometry of Phosphorylated and Sulfonated Peptides in Negative Ion Mode

NASA Astrophysics Data System (ADS)

Cook, Shannon L.; Jackson, Glen P.

2011-06-01

The dissociation behavior of phosphorylated and sulfonated peptide anions was explored using metastable atom-activated dissociation mass spectrometry (MAD-MS) and collision-induced dissociation (CID). A beam of high kinetic energy helium (He) metastable atoms was exposed to isolated phosphorylated and sulfonated peptides in the 3- and 2- charge states. Unlike CID, where phosphate losses are dominant, the major dissociation channels observed using MAD were Cα - C peptide backbone cleavages and neutral losses of CO2, H2O, and [CO2 + H2O] from the charge reduced (oxidized) product ion, consistent with an electron detachment dissociation (EDD) mechanism such as Penning ionization. Regardless of charge state or modification, MAD provides ample backbone cleavages with little modification loss, which allows for unambiguous PTM site determination. The relative abundance of certain fragment ions in MAD is also demonstrated to be somewhat sensitive to the number and location of deprotonation sites, with backbone cleavage somewhat favored adjacent to deprotonated sites like aspartic acid residues. MAD provides a complementary dissociation technique to CID, ECD, ETD, and EDD for peptide sequencing and modification identification. MAD offers the unique ability to analyze highly acidic peptides that contain few to no basic amino acids in either negative or positive ion mode.

Comparative analysis of effluent water quality from a municipal treatment plant and two on-site wastewater treatment systems.

PubMed

Garcia, Santos N; Clubbs, Rebekah L; Stanley, Jacob K; Scheffe, Brian; Yelderman, Joe C; Brooks, Bryan W

2013-06-01

Though decentralized on-site technologies are extensively employed for wastewater treatment around the globe, an understanding of effluent water quality impairments associated with these systems remain less understood than effluent discharges from centralized municipal wastewater treatment facilities. Using a unique experimental facility, a novel comparative analysis of effluent water quality was performed from model decentralized aerobic (ATS) and septic (STS) on-site wastewater treatment systems and a centralized municipal wastewater treatment plant (MTP). The ATS and STS units did not benefit from further soil treatment. Each system received common influent wastewater from the Waco, Texas, USA Metropolitan Area Regional Sewerage System. We tested the hypothesis that MTP effluent would exhibit higher water quality than on-site effluents, based on parameters selected for study. A tiered testing approach was employed to assess the three effluent discharges: select routine water quality parameters (Tier I), whole effluent toxicity (Tier II), and select endocrine-active compounds (Tier III). Contrary to our hypothesis, ATS effluent was not statistically different from MTP effluents, based on Tier I and III parameters, but reproductive responses of Daphnia magna were slightly more sensitive to ATS than MTP effluents. STS effluent water quality was identified as most degraded of the three wastewater treatment systems. Parameters used to assess centralized wastewater treatment plant effluent water quality such as whole effluent toxicity and endocrine active substances appear useful for water quality assessments of decentralized discharges. Aerobic on-site wastewater treatment systems may represent more robust options than traditional septic systems for on-site wastewater treatment in watersheds with appreciable groundwater - surface water exchange. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Minimal Replicator of Epstein-Barr Virus oriP

PubMed Central

Yates, John L.; Camiolo, Sarah M.; Bashaw, Jacqueline M.

2000-01-01

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports the replication and stable maintenance of plasmids in human cells. oriP contains two essential components, called the DS and the FR, both of which contain multiple binding sites for the EBV-encoded protein, EBNA-1. The DS appears to function as the replicator of oriP, while the FR acts in conjunction with EBNA-1 to prevent the loss of plasmids from proliferating cells. Because of EBNA-1's role in stabilizing plasmids through the FR, it has not been entirely clear to what extent EBNA-1 might be required for replication from oriP per se, and a recent study has questioned whether EBNA-1 has any direct role in replication. In the present study we found that plasmids carrying oriP required EBNA-1 to replicate efficiently even when assayed only 2 days after plasmids were introduced into the cell lines 143B and 293. Significantly, using 293 cells it was demonstrated that the plasmid-retention function of EBNA-1 and the FR did not contribute significantly to the accumulation of replicated plasmids, and the DS supported efficient EBNA-1-dependent replication in the absence of the FR. The DS contains two pairs of closely spaced EBNA-1 binding sites, and a previous study had shown that both sites within either pair are required for activity. However, it was unclear from previous work what additional sequences within the DS might be required. We found that each “half” of the DS, including a pair of closely spaced EBNA-1 binding sites, had significant replicator activity when the other half had been deleted. The only significant DNA sequences that the two halves of the DS share in common, other than EBNA-1 binding sites, is a 9-bp sequence that is present twice in the “left half” and once in the “right half.” These nonamer repeats, while not essential for activity, contributed significantly to the activity of each half of the DS. Two thymines occur at unique positions within EBNA-1 binding sites 1 and 4 at the DS and become sensitive to oxidation by permanganate when EBNA-1 binds, but mutation of each to the consensus base, adenine, actually improved the activity of each half of the DS slightly. In conclusion, the DS of oriP is an EBNA-1-dependent replicator, and its minimal active core appears to be simply two properly spaced EBNA-1 binding sites. PMID:10775587

A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins1[W

PubMed Central

Campos, Bruna Medéia; Sforça, Mauricio Luis; Ambrosio, Andre Luis Berteli; Domingues, Mariane Noronha; Brasil de Souza, Tatiana de Arruda Campos; Barbosa, João Alexandre Ribeiro Gonçalvez; Leme, Adriana Franco Paes; Perez, Carlos Alberto; Whittaker, Sara Britt-Marie; Murakami, Mario Tyago; Zeri, Ana Carolina de Matos; Benedetti, Celso Eduardo

2013-01-01

The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism. PMID:23709667

Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi.

PubMed

Liu, Jingwen; Cai, Weicong; Fang, Xian; Wang, Xueting; Li, Guiling

2018-04-01

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase.

PubMed

Debler, Erik W; Jain, Kanishk; Warmack, Rebeccah A; Feng, You; Clarke, Steven G; Blobel, Günter; Stavropoulos, Pete

2016-02-23

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

Raising Awareness of Individual Creative Potential in Bioscientists Using a Web-Site Based Approach

ERIC Educational Resources Information Center

Adams, David J.; Hugh-Jones, Siobhan; Sutherland, Ed

2010-01-01

We report the preliminary results of work with a unique, web-site-based approach designed to help individual bioscientists identify and develop their individual creative capacity. The site includes a number of features that encourage individuals to interact with creativity techniques, communicate with colleagues remotely using an electronic notice…

Blue Ribbon Web Sites Contest Winners.

ERIC Educational Resources Information Center

Southworth, Samuel A.

2001-01-01

Presents a collection of prize-winning Web sites created by K-8 teachers nationwide. Some of the unique features of the Web sites include an online student-written newspaper; a sing-along section; a chronicle of the past 3 years of classes to see how the classes have evolved; and student art and writing projects. (SM)

Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen*

PubMed Central

Kozlov, Sergei V.; Waardenberg, Ashley J.; Engholm-Keller, Kasper; Arthur, Jonathan W.; Graham, Mark E.; Lavin, Martin

2016-01-01

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM-dependence for translocation from the cytoplasm to the nucleus. These data provide new insights into the activation of ATM by oxidative stress through identification of novel substrates for ATM in the cytoplasm. PMID:26699800

The Role of Inhibitory Control in Behavioral and Physiological Expressions of Toddler Executive Function

PubMed Central

Morasch, Katherine C.; Bell, Martha Ann

2010-01-01

Eighty-one toddlers (ranging from 24 to 27 months) participated in a biobehavioral investigation of inhibitory control. Maternal-report measures of inhibitory control were related to laboratory tasks assessing inhibitory abilities under conditions of conflict, delay, and compliance challenge as well as toddler verbal ability. Additionally, unique variance in inhibitory control was explained by task-related changes in brain electrical activity at lateral frontal scalp sites as well as concurrent inhibitory task performance. Implications regarding neural correlates of executive function in early development and a central, organizing role of inhibitory processing in toddlerhood are discussed. PMID:20719337

Structural analysis of hierarchically organized zeolites

PubMed Central

Mitchell, Sharon; Pinar, Ana B.; Kenvin, Jeffrey; Crivelli, Paolo; Kärger, Jörg; Pérez-Ramírez, Javier

2015-01-01

Advances in materials synthesis bring about many opportunities for technological applications, but are often accompanied by unprecedented complexity. This is clearly illustrated by the case of hierarchically organized zeolite catalysts, a class of crystalline microporous solids that has been revolutionized by the engineering of multilevel pore architectures, which combine unique chemical functionality with efficient molecular transport. Three key attributes, the crystal, the pore and the active site structure, can be expected to dominate the design process. This review examines the adequacy of the palette of techniques applied to characterize these distinguishing features and their catalytic impact. PMID:26482337

Completely non-destructive elemental analysis of bulky samples by PGAA

NASA Astrophysics Data System (ADS)

Oura, Y.; Nakahara, H.; Sueki, K.; Sato, W.; Saito, A.; Tomizawa, T.; Nishikawa, T.

1999-01-01

NBAA (neutron beam activation analysis), which is a combination of PGAA and INAA by a single neutron irradiation, using an internal monostandard method is proposed as a very unique and promising method for the elemental analysis of voluminous and invaluable archaeological samples which do not allow even a scrape of the surface. It was applied to chinawares, Sueki ware, and bronze mirrors, and proved to be a very effective method for nondestructive analysis of not only major elements but also some minor elements such as boron that help solve archaeological problems of ears and sites of their production.

Disulfide-Bridged (Mo3S11) Cluster Polymer: Molecular Dynamics and Application as Electrode Material for a Rechargeable Magnesium Battery.

PubMed

Truong, Quang Duc; Kempaiah Devaraju, Murukanahally; Nguyen, Duc N; Gambe, Yoshiyuki; Nayuki, Keiichiro; Sasaki, Yoshikazu; Tran, Phong D; Honma, Itaru

2016-09-14

Exploring novel electrode materials is critical for the development of a next-generation rechargeable magnesium battery with high volumetric capacity. Here, we showed that a distinct amorphous molybdenum sulfide, being a coordination polymer of disulfide-bridged (Mo3S11) clusters, has great potential as a rechargeable magnesium battery cathode. This material provided good reversible capacity, attributed to its unique structure with high flexibility and capability of deformation upon Mg insertion. Free-terminal disulfide moiety may act as the active site for reversible insertion and extraction of magnesium.

Electronic structure contributions to reactivity in xanthine oxidase family enzymes.

PubMed

Stein, Benjamin W; Kirk, Martin L

2015-03-01

We review the xanthine oxidase (XO) family of pyranopterin molybdenum enzymes with a specific emphasis on electronic structure contributions to reactivity. In addition to xanthine and aldehyde oxidoreductases, which catalyze the two-electron oxidation of aromatic heterocycles and aldehyde substrates, this mini-review highlights recent work on the closely related carbon monoxide dehydrogenase (CODH) that catalyzes the oxidation of CO using a unique Mo-Cu heterobimetallic active site. A primary focus of this mini-review relates to how spectroscopy and computational methods have been used to develop an understanding of critical relationships between geometric structure, electronic structure, and catalytic function.

Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

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

Thal, David M.; Homan, Kristoff T.; Chen, Jun

2012-08-10

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. In this paper we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice withmore » paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.« less

Electron Transfer Mechanisms of DNA Repair by Photolyase

NASA Astrophysics Data System (ADS)

Zhong, Dongping

2015-04-01

Photolyase is a flavin photoenzyme that repairs two DNA base damage products induced by ultraviolet (UV) light: cyclobutane pyrimidine dimers and 6-4 photoproducts. With femtosecond spectroscopy and site-directed mutagenesis, investigators have recently made significant advances in our understanding of UV-damaged DNA repair, and the entire enzymatic dynamics can now be mapped out in real time. For dimer repair, six elementary steps have been characterized, including three electron transfer reactions and two bond-breaking processes, and their reaction times have been determined. A unique electron-tunneling pathway was identified, and the critical residues in modulating the repair function at the active site were determined. The dynamic synergy between the elementary reactions for maintaining high repair efficiency was elucidated, and the biological nature of the flavin active state was uncovered. For 6-4 photoproduct repair, a proton-coupled electron transfer repair mechanism has been revealed. The elucidation of electron transfer mechanisms and two repair photocycles is significant and provides a molecular basis for future practical applications, such as in rational drug design for curing skin cancer.

Immunoglobulin Gene Insertions and Deletions in the Affinity Maturation of HIV-1 Broadly Reactive Neutralizing Antibodies

PubMed Central

Kepler, Thomas B.; Liao, Hua-Xin; Alam, S. Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E.; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S. Abdool; Cohen, Myron S.; Walter, Emmanuel; Moody, M. Anthony; Wu, Xueling; Altae-Tran, Han R.; Georgiev, Ivelin S.; Kwong, Peter D.; Boyd, Scott D.; Fire, Andrew Z.; Mascola, John R.; Haynes, Barton F.

2014-01-01

Summary Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point-mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events. PMID:25211073

Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity.

PubMed

Kouno, Takahide; Silvas, Tania V; Hilbert, Brendan J; Shandilya, Shivender M D; Bohn, Markus F; Kelch, Brian A; Royer, William E; Somasundaran, Mohan; Kurt Yilmaz, Nese; Matsuo, Hiroshi; Schiffer, Celia A

2017-04-28

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5'-3' directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.

Access and Binding of H2S to Hemeproteins: The Case of HbI of Lucina pectinata.

PubMed

Boubeta, Fernando M; Bari, Sara E; Estrin, Dario A; Boechi, Leonardo

2016-09-15

Hydrogen sulfide (H2S) was recently discovered as a gasotransmitter, capable of coordinating to the heme iron of hemeproteins. H2S is unique for its ability to render varying concentrations of the nucleophilic conjugate bases (HS(-) or S(2-)), either as free or bound species with expected outcomes on its further reactivity. There is no direct evidence about which species (H2S, HS(-), or S(2-)) coordinates to the iron. We performed computer simulations to address the migration and binding processes of H2S species to the hemoglobin I of Lucina pectinata, which exhibits the highest affinity for the substrate measured to date. We found that H2S is the most favorable species in the migration from the bulk to the active site, through an internal pathway of the protein. After the coordination of H2S, an array of clustered water molecules modifies the active site environment, and assists in the subsequent deprotonation of the ligand, forming Fe(III)-SH(-). The feasibility of the second deprotonation of the coordinated ligand is also discussed.

Allosteric communication between the pyridoxal 5'-phosphate (PLP) and heme sites in the H2S generator human cystathionine β-synthase.

PubMed

Yadav, Pramod Kumar; Xie, Peter; Banerjee, Ruma

2012-11-02

Human cystathionine β-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.

Soil microbial biomass, activity and community composition along altitudinal gradients in the High Arctic (Billefjorden, Svalbard)

NASA Astrophysics Data System (ADS)

Kotas, Petr; Šantrůčková, Hana; Elster, Josef; Kaštovská, Eva

2018-03-01

The unique and fragile High Arctic ecosystems are vulnerable to global climate warming. The elucidation of factors driving microbial distribution and activity in arctic soils is essential for a comprehensive understanding of ecosystem functioning and its response to environmental change. The goals of this study were to investigate microbial biomass and activity, microbial community structure (MCS), and their environmental controls in soils along three elevational transects in the coastal mountains of Billefjorden, central Svalbard. Soils from four different altitudes (25, 275, 525 and 765 m above sea level) were analyzed for a suite of characteristics including temperature regimes, organic matter content, base cation availability, moisture, pH, potential respiration, and microbial biomass and community structure using phospholipid fatty acids (PLFAs). We observed significant spatial heterogeneity of edaphic properties among transects, resulting in transect-specific effects of altitude on most soil parameters. We did not observe any clear elevation pattern in microbial biomass, and microbial activity revealed contrasting elevational patterns between transects. We found relatively large horizontal variability in MCS (i.e., between sites of corresponding elevation in different transects), mainly due to differences in the composition of bacterial PLFAs, but also a systematic altitudinal shift in MCS related to different habitat preferences of fungi and bacteria, which resulted in high fungi-to-bacteria ratios at the most elevated sites. The biological soil crusts on these most elevated, unvegetated sites can host microbial assemblages of a size and activity comparable to those of the arctic tundra ecosystem. The key environmental factors determining horizontal and vertical changes in soil microbial properties were soil pH, organic carbon content, soil moisture and Mg2+ availability.

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity*

PubMed Central

Ramírez-Escudero, Mercedes; Gimeno-Pérez, María; González, Beatriz; Linde, Dolores; Merdzo, Zoran; Fernández-Lobato, María; Sanz-Aparicio, Julia

2016-01-01

Xanthophyllomyces dendrorhous β-fructofuranosidase (XdINV)is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We present here its three-dimensional structure, which shows the expected bimodular arrangement and also a long extension of its C terminus that together with an N-linked glycan mediate the formation of an unusual dimer. The two active sites of the dimer are connected by a long crevice, which might indicate its potential ability to accommodate branched fructans. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The inactive D80A mutant was used to obtain complexes with relevant substrates and products, with their crystals structures showing at least four binding subsites at each active site. Moreover, two different positions are observed from subsite +2 depending on the substrate, and thus, a flexible loop (Glu-334–His-343) is essential in binding sucrose and β(2–1)-linked oligosaccharides. Conversely, β(2–6) and neo-type substrates are accommodated mainly by stacking to Trp-105, explaining the production of neokestose and the efficient fructosylating activity of XdINV on α-glucosides. The role of relevant residues has been investigated by mutagenesis and kinetics measurements, and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates. PMID:26823463

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity.

PubMed

Ramírez-Escudero, Mercedes; Gimeno-Pérez, María; González, Beatriz; Linde, Dolores; Merdzo, Zoran; Fernández-Lobato, María; Sanz-Aparicio, Julia

2016-03-25

Xanthophyllomyces dendrorhousβ-fructofuranosidase (XdINV)is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We present here its three-dimensional structure, which shows the expected bimodular arrangement and also a long extension of its C terminus that together with anN-linked glycan mediate the formation of an unusual dimer. The two active sites of the dimer are connected by a long crevice, which might indicate its potential ability to accommodate branched fructans. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The inactive D80A mutant was used to obtain complexes with relevant substrates and products, with their crystals structures showing at least four binding subsites at each active site. Moreover, two different positions are observed from subsite +2 depending on the substrate, and thus, a flexible loop (Glu-334-His-343) is essential in binding sucrose and β(2-1)-linked oligosaccharides. Conversely, β(2-6) and neo-type substrates are accommodated mainly by stacking to Trp-105, explaining the production of neokestose and the efficient fructosylating activity of XdINV on α-glucosides. The role of relevant residues has been investigated by mutagenesis and kinetics measurements, and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix

PubMed Central

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-01-01

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPBKKK−, a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of α4β1 and α4β7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB–GAG interaction in the chemokine-like activity of this protein. PMID:11867726

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.

PubMed

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-03-05

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPB(KKK-), a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of alpha4beta1 and alpha4beta7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB-GAG interaction in the chemokine-like activity of this protein.

p21-activated kinase inhibitors.

PubMed

Rudolph, Joachim; Crawford, James J; Hoeflich, Klaus P; Chernoff, Jonathan

2013-01-01

The p21-activated kinases (PAKs) are Ser/Thr kinases in the STE20 kinase family with important roles in regulating cytoskeletal organization, cell migration, and signaling. The PAK enzyme family comprises six members subdivided into two groups: Group I, represented by PAK1, 2, and 3, and Group II, represented by PAK 4, 5, and 6, based on sequence and structural homology. Individual PAK isoforms were found to be overexpressed and amplified in a variety of human cancers, and in vitro and in vivo studies using genetically engineered systems as well as small-molecule tool compounds have suggested therapeutic utility of PAKs as oncology targets. The identification of potent and kinome-selective ATP-competitive PAK inhibitors has proven challenging, likely caused by the openness and unique plasticity of the ATP-binding site of PAK enzymes. Progress in achieving increased kinase selectivity has been achieved with certain inhibitors but at the expense of increased molecular weight. Allosteric inhibitors, such as IPA-3, leverage the unique Group I PAK autoregulatory domain for selective inhibition, and this approach might provide an outlet to evade the kinase selectivity challenges observed with ATP-competitive PAK inhibitors. © 2013 Elsevier Inc. All rights reserved.

Ligand-specific Deactivation Time Course of GluN1/GluN2D NMDA Receptors

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

K Vance; N Simorowski; S Traynelis

2011-12-31

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors that mediate a majority of excitatory synaptic transmission. One unique property of GluN1/GluN2D NMDA receptors is an unusually prolonged deactivation time course following the removal of L-glutamate. Here we show, using x-ray crystallography and electrophysiology, that the deactivation time course of GluN1/GluN2D receptors is influenced by the conformational variability of the ligand-binding domain (LBD) as well as the structure of the activating ligand. L-glutamate and L-CCG-IV induce significantly slower deactivation time courses compared with other agonists. Crystal structures of the isolated GluN2D LBD in complex with various ligands revealmore » that the binding of L-glutamate induces a unique conformation at the backside of the ligand-binding site in proximity to the region at which the transmembrane domain would be located in the intact receptors. These data suggest that the activity of the GluN1/GluN2D NMDA receptor is controlled distinctively by the endogenous neurotransmitter L-glutamate.« less

Regioselective Reactions for Programmable Resveratrol Oligomer Synthesis

PubMed Central

Snyder, Scott A.; Gollner, Andreas; Chiriac, Maria I.

2011-01-01

Although much attention has been devoted to resveratrol, a unique polyphenol produced by plants throughout the world and credited as potentially being responsible for the so-called “French paradox” given its broad spectrum activity, the hundreds of oligomeric materials derived from it have been largely ignored despite their similarly high biochemical potential. Challenges in achieving their isolation in quantity from natural sources, coupled with an inability to rationally prepare them in the laboratory, are the main culprits. Here we show that a programmable, controlled, and potentially scaleable synthesis of the resveratrol family is possible through a unique three-stage design. These efforts required novel tactics coupled with strategy- and reagent-guided functionalizations to differentiate two distinct cores possessing multiple sites with the same and/or similar reactivity, ultimately leading to five higher-order natural products. We anticipate that this work 1) demonstrates that challenging, positionally-selective functionalizations of complex materials are possible where biosynthetic studies have indicated otherwise, 2) provides materials and tools to finally unlock the full biochemical potential of the family, particularly from the standpoint of activity and drug-property optimization, and 3) affords an intellectual framework to potentially access other oligomeric families controllably. PMID:21697944

Functional macrophages and gastrointestinal disorders.

PubMed

Liu, Yue-Hong; Ding, Yue; Gao, Chen-Chen; Li, Li-Sheng; Wang, Yue-Xiu; Xu, Jing-Dong

2018-03-21

Macrophages (MΦ) differentiate from blood monocytes and participate in innate and adaptive immunity. Because of their abilities to recognize pathogens and activate bactericidal activities, MΦ are always discovered at the site of immune defense. MΦ in the intestine are unique, such that in the healthy intestine, they possess complex mechanisms to protect the gut from inflammation. In these complex mechanisms, they produce anti-inflammatory cytokines, such as interleukin-10 and transforming growth factor-β, and inhibit the inflammatory pathways mediated by Toll-like receptors. It has been demonstrated that resident MΦ play a crucial role in maintaining intestinal homeostasis, and they can be recognized by their unique markers. Nonetheless, in the inflamed intestine, the function of MΦ will change because of environmental variation, which may be one of the mechanisms of inflammatory bowel disease (IBD). We provide further explanation about these mechanisms in our review. In addition, we review recent discoveries that MΦ may be involved in the development of gastrointestinal tumors. We will highlight the possible therapeutic targets for the management of IBD and gastrointestinal tumors, and we also discuss why more details are needed to fully understand all other effects of intestinal MΦ.

Early evidence (ca. 12,000 B.P.) for feasting at a burial cave in Israel

PubMed Central

Munro, Natalie D.; Grosman, Leore

2010-01-01

Feasting is one of humanity's most universal and unique social behaviors. Although evidence for feasting is common in the early agricultural societies of the Neolithic, evidence in pre-Neolithic contexts is more elusive. We found clear evidence for feasting on wild cattle and tortoises at Hilazon Tachtit cave, a Late Epipaleolithic (12,000 calibrated years B.P.) burial site in Israel. This includes unusually high densities of butchered tortoise and wild cattle remains in two structures, the unique location of the feasting activity in a burial cave, and the manufacture of two structures for burial and related feasting activities. The results indicate that community members coalesced at Hilazon to engage in special rituals to commemorate the burial of the dead and that feasts were central elements in these important events. Feasts likely served important roles in the negotiation and solidification of social relationships, the integration of communities, and the mitigation of scalar stress. These and other social changes in the Natufian period mark significant changes in human social complexity that continued into the Neolithic period. Together, social and economic change signal the very beginning of the agricultural transition. PMID:20805510

Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor*

PubMed Central

Kobayashi, Hidetomo; Utsunomiya, Hiroko; Yamanaka, Hiroyasu; Sei, Yoshihisa; Katunuma, Nobuhiko; Okamoto, Keinosuke; Tsuge, Hideaki

2009-01-01

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 Å resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs. PMID:19654332

Identification of a unique loss-of-function mutation in IGF1R and a crosstalk between IGF1R and Wnt/β-catenin signaling pathways.

PubMed

Jamwal, Gayatri; Singh, Gurjinder; Dar, Mohd Saleem; Singh, Paramjeet; Bano, Nasima; Syed, Sajad Hussain; Sandhu, Padmani; Akhter, Yusuf; Monga, Satdarshan P; Dar, Mohd Jamal

2018-06-01

IGF1R is a ubiquitous receptor tyrosine kinase that plays critical roles in cell proliferation, growth and survival. Clinical studies have demonstrated upregulation of IGF1R mediated signaling in a number of malignancies including colon, breast, and lung cancers. Overexpression of the IGF1R in these malignancies is associated with a poor prognosis and overall survival. IGF1R specific kinase inhibitors have failed in multiple clinical trials partly because of the complex nature of IGF1R signaling. Thus identifying new binding partners and allosteric sites on IGF1R are emerging areas of research. More recently, IGF1R has been shown to translocate into the nucleus and perform many functions. In this study, we generated a library of IGF1R deletion and point mutants to examine IGF1R subcellular localization and activation of downstream signaling pathways. We show that the nuclear localization of IGF1R is primarily defined by its cytoplasmic domain. We identified a cross-talk between IGF1R and Wnt/β-catenin signaling pathways and showed, for the first time, that IGF1R is associated with upregulation of TCF-mediated β-catenin transcriptional activity. Using loss-of-function mutants, deletion analysis and IGF1R specific inhibitor(s), we show that cytoplasmic and nuclear activities are two independent functions of IGF1R. Furthermore, we identified a unique loss-of-function mutation in IGF1R. This unique loss-of-function mutant retains only nuclear functions and sits in a pocket, outside ATP and substrate binding region, that is suited for designing allosteric inhibitors of IGF1R. Copyright © 2018 Elsevier B.V. All rights reserved.

The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates

PubMed Central

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

The Congolobe project, a multidisciplinary study of Congo deep-sea fan lobe complex: Overview of methods, strategies, observations and sampling

NASA Astrophysics Data System (ADS)

Rabouille, C.; Olu, K.; Baudin, F.; Khripounoff, A.; Dennielou, B.; Arnaud-Haond, S.; Babonneau, N.; Bayle, C.; Beckler, J.; Bessette, S.; Bombled, B.; Bourgeois, S.; Brandily, C.; Caprais, J. C.; Cathalot, C.; Charlier, K.; Corvaisier, R.; Croguennec, C.; Cruaud, P.; Decker, C.; Droz, L.; Gayet, N.; Godfroy, A.; Hourdez, S.; Le Bruchec, J.; Saout, J.; Le Saout, M.; Lesongeur, F.; Martinez, P.; Mejanelle, L.; Michalopoulos, P.; Mouchel, O.; Noel, P.; Pastor, L.; Picot, M.; Pignet, P.; Pozzato, L.; Pruski, A. M.; Rabiller, M.; Raimonet, M.; Ragueneau, O.; Reyss, J. L.; Rodier, P.; Ruesch, B.; Ruffine, L.; Savignac, F.; Senyarich, C.; Schnyder, J.; Sen, A.; Stetten, E.; Sun, Ming Yi; Taillefert, M.; Teixeira, S.; Tisnerat-Laborde, N.; Toffin, L.; Tourolle, J.; Toussaint, F.; Vétion, G.; Jouanneau, J. M.; Bez, M.; Congolobe Group:

2017-08-01

The presently active region of the Congo deep-sea fan (around 330,000 km2), called the terminal lobes or lobe complex, covers an area of 2500 km2 at 4700-5100 m water depth and 750-800 km offshore. It is a unique sedimentary area in the world ocean fed by a submarine canyon and a channel-levee system which presently deliver large amounts of organic carbon originating from the Congo River by turbidity currents. This particularity is due to the deep incision of the shelf by the Congo canyon, up to 30 km into the estuary, which funnels the Congo River sediments into the deep-sea. The connection between the river and the canyon is unique for major world rivers. In 2011, two cruises (WACS leg 2 and Congolobe) were conducted to simultaneously investigate the geology, organic and inorganic geochemistry, and micro- and macro-biology of the terminal lobes of the Congo deep-sea fan. Using this multidisciplinary approach, the morpho-sedimentary features of the lobes were characterized along with the origin and reactivity of organic matter, the recycling and burial of biogenic compounds, the diversity and function of bacterial and archaeal communities within the sediment, and the biodiversity and functioning of the faunal assemblages on the seafloor. Six different sites were selected for this study: Four distributed along the active channel from the lobe complex entrance to the outer rim of the sediment deposition zone, and two positioned cross-axis and at increasing distance from the active channel, thus providing a gradient in turbidite particle delivery and sediment age. This paper aims to provide the general context of this multidisciplinary study. It describes the general features of the site and the overall sampling strategy and provides the initial habitat observations to guide the other in-depth investigations presented in this special issue. Detailed bathymetry of each sampling site using 0.1-1 m resolution multibeam obtained with a remotely operated vehicle (ROV) shows progressive widening and smoothing of the channel-levees with increasing depth and reveals a complex morphology with channel bifurcations, erosional features and massive deposits. Dense ecosystems surveyed in the study area gather high density clusters of two large-sized species of symbiotic Vesicomyidae bivalves and microbial mats. These assemblages, which are rarely observed in sedimentary zones, resemble those based on chemosynthesis at cold-seep sites, such as the active pockmarks encountered along the Congo margin, and share with these sites the dominant vesicomyid species Christineconcha regab. Sedimentation rates estimated in the lobe complex range between 0.5 and 10 cm yr-1, which is 2-3 orders of magnitude higher than values generally encountered at abyssal depths. The bathymetry, faunal assemblages and sedimentation rates make the Congo lobe complex a highly peculiar deep-sea habitat driven by high inputs of terrigenous material delivered by the Congo channel-levee system.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-08-15

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed Central

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2. PMID:9693111

In situ characterization of cofacial Co(IV) centers in Co4O4 cubane: Modeling the high-valent active site in oxygen-evolving catalysts.

PubMed

Brodsky, Casey N; Hadt, Ryan G; Hayes, Dugan; Reinhart, Benjamin J; Li, Nancy; Chen, Lin X; Nocera, Daniel G

2017-04-11

The Co 4 O 4 cubane is a representative structural model of oxidic cobalt oxygen-evolving catalysts (Co-OECs). The Co-OECs are active when residing at two oxidation levels above an all-Co(III) resting state. This doubly oxidized Co(IV) 2 state may be captured in a Co(III) 2 (IV) 2 cubane. We demonstrate that the Co(III) 2 (IV) 2 cubane may be electrochemically generated and the electronic properties of this unique high-valent state may be probed by in situ spectroscopy. Intervalence charge-transfer (IVCT) bands in the near-IR are observed for the Co(III) 2 (IV) 2 cubane, and spectroscopic analysis together with electrochemical kinetics measurements reveal a larger reorganization energy and a smaller electron transfer rate constant for the doubly versus singly oxidized cubane. Spectroelectrochemical X-ray absorption data further reveal systematic spectral changes with successive oxidations from the cubane resting state. Electronic structure calculations correlated to experimental data suggest that this state is best represented as a localized, antiferromagnetically coupled Co(IV) 2 dimer. The exchange coupling in the cofacial Co(IV) 2 site allows for parallels to be drawn between the electronic structure of the Co 4 O 4 cubane model system and the high-valent active site of the Co-OEC, with specific emphasis on the manifestation of a doubly oxidized Co(IV) 2 center on O-O bond formation.

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism

PubMed Central

Kim, Seonah; Ståhlberg, Jerry; Sandgren, Mats; Paton, Robert S.; Beckham, Gregg T.

2014-01-01

Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η1-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η1) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds. PMID:24344312

In situ characterization of cofacial Co(IV) centers in Co 4O 4 cubane: Modeling the high-valent active site in oxygen-evolving catalysts

DOE PAGES

Brodsky, Casey N.; Hadt, Ryan G.; Hayes, Dugan; ...

2017-03-27

The Co 4O 4 cubane is a representative structural model of oxidic cobalt oxygen evolving catalysts (Co-OECs). The Co-OECs are active when residing at two oxidation levels above an all Co(III) resting state. This doubly oxidized Co(IV) 2 state may be captured in a Co(III) 2(IV) 2 cubane. We demonstrate that the Co(III) 2(IV) 2 cubane may be electrochemically generated and the electronic properties of this unique high-valent state may be probed by in situ spectroscopy. Intervalence charge transfer (IVCT) bands in the near-IR are observed for the Co(III) 2(IV) 2 cubane, and spectroscopic analysis together with electrochemical kinetics measurementsmore » reveal a larger reorganization energy and a smaller electron transfer rate constant for the doubly versus singly oxidized cubane. Spectroelectrochemical X-ray absorption data further reveal systematic spectral changes with successive oxidations from the cubane resting state. Electronic structure calculations correlated to experimental data suggest that this state is best represented as a localized, antiferromagnetically coupled Co(IV) 2 dimer. The exchange coupling in the cofacial Co(IV) 2 site allows for parallels to be drawn between the electronic structure of the Co 4O 4 cubane model system and the high valent active site of the Co-OEC, with specific emphasis on the manifestation of a doubly oxidized Co(IV) 2 center on O–O bond formation.« less

Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.

PubMed

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.

Disrupting the brain to validate hypotheses on the neurobiology of language

PubMed Central

Papeo, Liuba; Pascual-Leone, Alvaro; Caramazza, Alfonso

2013-01-01

Comprehension of words is an important part of the language faculty, involving the joint activity of frontal and temporo-parietal brain regions. Transcranial Magnetic Stimulation (TMS) enables the controlled perturbation of brain activity, and thus offers a unique tool to test specific predictions about the causal relationship between brain regions and language understanding. This potential has been exploited to better define the role of regions that are classically accepted as part of the language-semantic network. For instance, TMS has contributed to establish the semantic relevance of the left anterior temporal lobe, or to solve the ambiguity between the semantic vs. phonological function assigned to the left inferior frontal gyrus (LIFG). We consider, more closely, the results from studies where the same technique, similar paradigms (lexical-semantic tasks) and materials (words) have been used to assess the relevance of regions outside the classically-defined language-semantic network—i.e., precentral motor regions—for the semantic analysis of words. This research shows that different aspects of the left precentral gyrus (primary motor and premotor sites) are sensitive to the action-non action distinction of words' meanings. However, the behavioral changes due to TMS over these sites are incongruent with what is expected after perturbation of a task-relevant brain region. Thus, the relationship between motor activity and language-semantic behavior remains far from clear. A better understanding of this issue could be guaranteed by investigating functional interactions between motor sites and semantically-relevant regions. PMID:23630480

Ferritin: the protein nanocage and iron biomineral in health and in disease.

PubMed

Theil, Elizabeth C

2013-11-04

At the center of iron and oxidant metabolism is the ferritin superfamily: protein cages with Fe(2+) ion channels and two catalytic Fe/O redox centers that initiate the formation of caged Fe2O3·H2O. Ferritin nanominerals, initiated within the protein cage, grow inside the cage cavity (5 or 8 nm in diameter). Ferritins contribute to normal iron flow, maintenance of iron concentrates for iron cofactor syntheses, sequestration of iron from invading pathogens, oxidant protection, oxidative stress recovery, and, in diseases where iron accumulates excessively, iron chelation strategies. In eukaryotic ferritins, biomineral order/crystallinity is influenced by nucleation channels between active sites and the mineral growth cavity. Animal ferritin cages contain, uniquely, mixtures of catalytically active (H) and inactive (L) polypeptide subunits with varied rates of Fe(2+)/O2 catalysis and mineral crystallinity. The relatively low mineral order in liver ferritin, for example, coincides with a high percentage of L subunits and, thus, a low percentage of catalytic sites and nucleation channels. Low mineral order facilitates rapid iron turnover and the physiological role of liver ferritin as a general iron source for other tissues. Here, current concepts of ferritin structure/function/genetic regulation are discussed and related to possible therapeutic targets such as mini-ferritin/Dps protein active sites (selective pathogen inhibition in infection), nanocage pores (iron chelation in therapeutic hypertransfusion), mRNA noncoding, IRE riboregulator (normalizing the ferritin iron content after therapeutic hypertransfusion), and protein nanovessels to deliver medicinal or sensor cargo.

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

Single Atomic Iron Catalysts for Oxygen Reduction in Acidic Media: Particle Size Control and Thermal Activation

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

Zhang, Hanguang; Hwang, Sooyeon; Wang, Maoyu

It remains a grand challenge to replace platinum group metal (PGM) catalysts with earth-abundant materials for the oxygen reduction reaction (ORR) in acidic media, which is crucial for large-scale deployment of proton exchange membrane fuel cells (PEMFCs). Here, we report a high-performance atomic Fe catalyst derived from chemically Fe-doped zeolitic imidazolate frameworks (ZIFs) by directly bonding Fe ions to imidazolate ligands within 3D frameworks. Although the ZIF was identified as a promising precursor, the new synthetic chemistry enables the creation of well-dispersed atomic Fe sites embedded into porous carbon without the formation of aggregates. The size of catalyst particles ismore » tunable through synthesizing Fe-doped ZIF nanocrystal precursors in a wide range from 20 to 1000 nm followed by one-step thermal activation. Similar to Pt nanoparticles, the unique size control without altering chemical properties afforded by this approach is able to increase the number of PGM-free active sites. The best ORR activity is measured with the catalyst at a size of 50 nm. Further size reduction to 20 nm leads to significant particle agglomeration, thus decreasing the activity. Using the homogeneous atomic Fe model catalysts, we elucidated the active site formation process through correlating measured ORR activity with the change of chemical bonds in precursors during thermal activation up to 1100 °C. The critical temperature to form active sites is 800 °C, which is associated with a new Fe species with a reduced oxidation number (from Fe3+ to Fe2+) likely bonded with pyridinic N (FeN4) embedded into the carbon planes. Further increasing the temperature leads to continuously enhanced activity, linked to the rise of graphitic N and Fe–N species. The new atomic Fe catalyst has achieved respectable ORR activity in challenging acidic media (0.5 M H2SO4), showing a half-wave potential of 0.85 V vs RHE and leaving only a 30 mV gap with Pt/C (60 μgPt/cm2). Enhanced stability is attained with the same catalyst, which loses only 20 mV after 10 000 potential cycles (0.6–1.0 V) in O2 saturated acid. The high-performance atomic Fe PGM-free catalyst holds great promise as a replacement for Pt in future PEMFCs.« less

Single Atomic Iron Catalysts for Oxygen Reduction in Acidic Media: Particle Size Control and Thermal Activation

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

Zhang, Hanguang; Hwang, Sooyeon; Wang, Maoyu

It remains a grand challenge to replace platinum group metal (PGM) catalysts with earth-abundant materials for the oxygen reduction reaction (ORR) in acidic media, which is crucial for large-scale deployment of proton exchange membrane fuel cells (PEMFCs). We report a high-performance atomic Fe catalyst derived from chemically Fe-doped zeolitic imidazolate frameworks (ZIFs) by directly bonding Fe ions to imidazolate ligands within 3D frameworks. Although the ZIF was identified as a promising precursor, the new synthetic chemistry enables the creation of well-dispersed atomic Fe sites embedded into porous carbon without the formation of aggregates. The size of catalyst particles is tunablemore » through synthesizing Fe-doped ZIF nanocrystal precursors in a wide range from 20 to 1000 nm followed by one-step thermal activation. Similar to Pt nanoparticles, the unique size control without altering chemical properties afforded by this approach is able to increase the number of PGM-free active sites. The best ORR activity is measured with the catalyst at a size of 50 nm. Further size reduction to 20 nm leads to significant particle agglomeration, thus decreasing the activity. In using the homogeneous atomic Fe model catalysts, we elucidated the active site formation process through correlating measured ORR activity with the change of chemical bonds in precursors during thermal activation up to 1100 °C. The critical temperature to form active sites is 800 °C, which is associated with a new Fe species with a reduced oxidation number (from Fe 3+ to Fe 2+) likely bonded with pyridinic N (FeN 4) embedded into the carbon planes. Further increasing the temperature leads to continuously enhanced activity, linked to the rise of graphitic N and Fe–N species. The new atomic Fe catalyst has achieved respectable ORR activity in challenging acidic media (0.5 M H 2SO 4), showing a half-wave potential of 0.85 V vs RHE and leaving only a 30 mV gap with Pt/C (60 μg Pt/cm 2). Finally, enhanced stability is attained with the same catalyst, which loses only 20 mV after 10 000 potential cycles (0.6–1.0 V) in O 2 saturated acid. The high-performance atomic Fe PGM-free catalyst holds great promise as a replacement for Pt in future PEMFCs.« less

Single Atomic Iron Catalysts for Oxygen Reduction in Acidic Media: Particle Size Control and Thermal Activation

DOE PAGES

Zhang, Hanguang; Hwang, Sooyeon; Wang, Maoyu; ...

2017-09-13

It remains a grand challenge to replace platinum group metal (PGM) catalysts with earth-abundant materials for the oxygen reduction reaction (ORR) in acidic media, which is crucial for large-scale deployment of proton exchange membrane fuel cells (PEMFCs). We report a high-performance atomic Fe catalyst derived from chemically Fe-doped zeolitic imidazolate frameworks (ZIFs) by directly bonding Fe ions to imidazolate ligands within 3D frameworks. Although the ZIF was identified as a promising precursor, the new synthetic chemistry enables the creation of well-dispersed atomic Fe sites embedded into porous carbon without the formation of aggregates. The size of catalyst particles is tunablemore » through synthesizing Fe-doped ZIF nanocrystal precursors in a wide range from 20 to 1000 nm followed by one-step thermal activation. Similar to Pt nanoparticles, the unique size control without altering chemical properties afforded by this approach is able to increase the number of PGM-free active sites. The best ORR activity is measured with the catalyst at a size of 50 nm. Further size reduction to 20 nm leads to significant particle agglomeration, thus decreasing the activity. In using the homogeneous atomic Fe model catalysts, we elucidated the active site formation process through correlating measured ORR activity with the change of chemical bonds in precursors during thermal activation up to 1100 °C. The critical temperature to form active sites is 800 °C, which is associated with a new Fe species with a reduced oxidation number (from Fe 3+ to Fe 2+) likely bonded with pyridinic N (FeN 4) embedded into the carbon planes. Further increasing the temperature leads to continuously enhanced activity, linked to the rise of graphitic N and Fe–N species. The new atomic Fe catalyst has achieved respectable ORR activity in challenging acidic media (0.5 M H 2SO 4), showing a half-wave potential of 0.85 V vs RHE and leaving only a 30 mV gap with Pt/C (60 μg Pt/cm 2). Finally, enhanced stability is attained with the same catalyst, which loses only 20 mV after 10 000 potential cycles (0.6–1.0 V) in O 2 saturated acid. The high-performance atomic Fe PGM-free catalyst holds great promise as a replacement for Pt in future PEMFCs.« less

Soil-plant-microbial relations in hydrothermally altered soils of Northern California

USGS Publications Warehouse

Blecker, S.W.; Stillings, L.L.; DeCrappeo, N.M.; Ippolito, J.A.

2014-01-01

Soils developed on relict hydrothermally altered soils throughout the Western USA present unique opportunities to study the role of geology on above and belowground biotic activity and composition. Soil and vegetation samples were taken at three unaltered andesite and three hydrothermally altered (acid-sulfate) sites located in and around Lassen VolcanicNational Park in northeastern California. In addition, three different types of disturbed areas (clearcut, thinned, and pipeline) were sampled in acid-sulfate altered sites. Soils were sampled (0–15 cm) in mid-summer 2010 from both under-canopy and between-canopy areas within each of the sites. Soils were analyzed for numerous physical and chemical properties along with soil enzyme assays, C and N mineralization potential, microbial biomass-C and C-substrate utilization. Field vegetation measurements consisted of canopy cover by life form (tree, shrub, forb, and grass), tree and shrub density, and above-ground net primary productivity of the understory. Overall, parameters at the clearcut sites were more similar to the unaltered sites, while parameters at the thinned and pipeline sites were more similar to the altered sites. We employed principal components analysis (PCA) to develop two soil quality indices (SQI) to help quantify the differences among the sites: one based on the correlation between soil parameters and canopy cover, and the second based on six sub-indices. Soil quality indices developed in these systems could provide a means for monitoring and identifying key relations between the vegetation, soils, and microorganisms.

Accumulation of slightly deleterious mutations in the mitochondrial genome: a hallmark of animal domestication.

PubMed

Hughes, Austin L

2013-02-15

The hypothesis that domestication leads to a relaxation of purifying selection on mitochondrial (mt) genomes was tested by comparative analysis of mt genes from dog, pig, chicken, and silkworm. The three vertebrate species showed mt genome phylogenies in which domestic and wild isolates were intermingled, whereas the domestic silkworm (Bombyx mori) formed a distinct cluster nested within its closest wild relative (Bombyx mandarina). In spite of these differences in phylogenetic pattern, significantly greater proportions of nonsynonymous SNPs than of synonymous SNPs were unique to the domestic populations of all four species. Likewise, in all four species, significantly greater proportions of RNA-encoding SNPs than of synonymous SNPs were unique to the domestic populations. Thus, domestic populations were characterized by an excess of unique polymorphisms in two categories generally subject to purifying selection: nonsynonymous sites and RNA-encoding sites. Many of these unique polymorphisms thus seem likely to be slightly deleterious; the latter hypothesis was supported by the generally lower gene diversities of polymorphisms unique to domestic populations in comparison to those of polymorphisms shared by domestic and wild populations. Copyright © 2012 Elsevier B.V. All rights reserved.

Activation of Escherichia coli antiterminator BglG requires its phosphorylation

PubMed Central

Rothe, Fabian M.; Bahr, Thomas; Stülke, Jörg; Rak, Bodo; Görke, Boris

2012-01-01

Transcriptional antiterminator proteins of the BglG family control the expression of enzyme II (EII) carbohydrate transporters of the bacterial phosphotransferase system (PTS). In the PTS, phosphoryl groups are transferred from phosphoenolpyruvate (PEP) via the phosphotransferases enzyme I (EI) and HPr to the EIIs, which phosphorylate their substrates during transport. Activity of the antiterminators is negatively controlled by reversible phosphorylation catalyzed by the cognate EIIs in response to substrate availability and positively controlled by the PTS. For the Escherichia coli BglG antiterminator, two different mechanisms for activation by the PTS were proposed. According to the first model, BglG is activated by HPr-catalyzed phosphorylation at a site distinct from the EII-dependent phosphorylation site. According to the second model, BglG is not activated by phosphorylation, but solely through interaction with EI and HPr, which are localized at the cell pole. Subsequently BglG is released from the cell pole to the cytoplasm as an active dimer. Here we addressed this discrepancy and found that activation of BglG requires phosphorylatable HPr or the HPr homolog FruB in vivo. Further, we uniquely demonstrate that purified BglG protein becomes phosphorylated by FruB as well as by HPr in vitro. Histidine residue 208 in BglG is essential for this phosphorylation. These data suggest that BglG is in fact activated by phosphorylation and that there is no principal difference between the PTS-exerted mechanisms controlling the activities of BglG family proteins in Gram-positive and Gram-negative bacteria. PMID:22984181

Selectivity of externally facing ion-binding sites in the Na/K pump to alkali metals and organic cations

PubMed Central

Ratheal, Ian M.; Virgin, Gail K.; Yu, Haibo; Roux, Benoît; Gatto, Craig; Artigas, Pablo

2010-01-01

The Na/K pump is a P-type ATPase that exchanges three intracellular Na+ ions for two extracellular K+ ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either Na+ or K+; site III binds only Na+) are poorly understood. We studied cation selectivity by outward-facing sites (high K+ affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium+, methylguanidinium+, and aminoguanidinium+ produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating K+, and (ii) induction of pump-mediated, guanidinium-derivative–carried inward current at negative potentials without Na+ and K+. In contrast, formamidinium+ and acetamidinium+ induced K+-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K+ congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only Li+ induced Na+-like VDI, whereas all metals tested except Na+ induced K+-like outward currents. Pump-mediated K+-like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium+ derivatives suggest that Na+ binds to site III in a hydrated form and that the inward current observed without external Na+ and K+ represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites. PMID:20937860

U1snRNP-mediated suppression of polyadenylation in conjunction with the RNA structure controls poly (A) site selection in foamy viruses

PubMed Central

2013-01-01

Background During reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5′LTR and activate polyadenylation in the 3′LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal. Results Here, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5′LTR. In contrast, polyadenylation at the 3′LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation. Conclusion Recently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5’end of their RNA. At the 3’end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression. PMID:23718736

Mn(II) Oxidation by the Multicopper Oxidase Complex Mnx: A Coordinated Two-Stage Mn(II)/(III) and Mn(III)/(IV) Mechanism.

PubMed

Soldatova, Alexandra V; Romano, Christine A; Tao, Lizhi; Stich, Troy A; Casey, William H; Britt, R David; Tebo, Bradley M; Spiro, Thomas G

2017-08-23

The bacterial manganese oxidase MnxG of the Mnx protein complex is unique among multicopper oxidases (MCOs) in carrying out a two-electron metal oxidation, converting Mn(II) to MnO 2 nanoparticles. The reaction occurs in two stages: Mn(II) → Mn(III) and Mn(III) → MnO 2 . In a companion study , we show that the electron transfer from Mn(II) to the low-potential type 1 Cu of MnxG requires an activation step, likely forming a hydroxide bridge at a dinuclear Mn(II) site. Here we study the second oxidation step, using pyrophosphate (PP) as a Mn(III) trap. PP chelates Mn(III) produced by the enzyme and subsequently allows it to become a substrate for the second stage of the reaction. EPR spectroscopy confirms the presence of Mn(III) bound to the enzyme. The Mn(III) oxidation step does not involve direct electron transfer to the enzyme from Mn(III), which is shown by kinetic measurements to be excluded from the Mn(II) binding site. Instead, Mn(III) is proposed to disproportionate at an adjacent polynuclear site, thereby allowing indirect oxidation to Mn(IV) and recycling of Mn(II). PP plays a multifaceted role, slowing the reaction by complexing both Mn(II) and Mn(III) in solution, and also inhibiting catalysis, likely through binding at or near the active site. An overall mechanism for Mnx-catalyzed MnO 2 production from Mn(II) is presented.

Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.

PubMed

Gay, Sean C; Shah, Manish B; Talakad, Jyothi C; Maekawa, Keiko; Roberts, Arthur G; Wilderman, P Ross; Sun, Ling; Yang, Jane Y; Huelga, Stephanie C; Hong, Wen-Xu; Zhang, Qinghai; Stout, C David; Halpert, James R

2010-04-01

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

ERK2-mediated C-terminal serine phosphorylation of p300 is vital to the regulation of epidermal growth factor-induced keratin 16 gene expression.

PubMed

Chen, Yun-Ju; Wang, Ying-Nai; Chang, Wen-Chang

2007-09-14

We previously reported that the epidermal growth factor (EGF) regulates the gene expression of keratin 16 by activating the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling which in turn enhances the recruitment of p300 to the keratin 16 promoter. The recruited p300 functionally cooperates with Sp1 and c-Jun to regulate the gene expression of keratin 16. This study investigated in detail the molecular events incurred upon p300 whereby EGF caused an enhanced interaction between p300 and Sp1. EGF apparently induced time- and dose-dependent phosphorylation of p300, both in vitro and in vivo, through the activation of ERK2. The six potential ERK2 phosphorylation sites, including three threonine and three serine residues as revealed by sequential analysis, were first identified in vitro. Confirmation of these six sites in vivo indicated that these three serine residues (Ser-2279, Ser-2315, and Ser-2366) on the C terminus of p300 were the major signaling targets of EGF. Furthermore, the C-terminal serine phosphorylation of p300 stimulated its histone acetyltransferase activity and enhanced its interaction with Sp1. These serine phosphorylation sites on p300 controlled the p300 recruitment to the keratin 16 promoter. When all three serine residues on p300 were replaced by alanine, EGF could no longer induce the gene expression of keratin 16. Taken together, these results strongly suggested that the ERK2-mediated C-terminal serine phosphorylation of p300 was a key event in the regulation of EGF-induced keratin 16 expression. These results also constituted the first report identifying the unique p300 phosphorylation sites induced by ERK2 in vivo.

Registry-linked electronic influenza vaccine provider reminders: a cluster-crossover trial.

PubMed

Stockwell, Melissa S; Catallozzi, Marina; Camargo, Stewin; Ramakrishnan, Rajasekhar; Holleran, Stephen; Findley, Sally E; Kukafka, Rita; Hofstetter, Annika M; Fernandez, Nadira; Vawdrey, David K

2015-01-01

To determine the impact of a vaccination reminder in an electronic health record supplemented with data from an immunization information system (IIS). A noninterruptive influenza vaccination reminder, based on a real-time query of hospital and city IIS, was used at 4 urban, academically affiliated clinics serving a low-income population. Using a randomized cluster-crossover design, each study site had "on" and "off" period during the fall and winter of 2011-2012. Influenza vaccination during a clinic visit was assessed for 6-month to 17-year-old patients. To assess sustainability, the reminder was active at all sites during the 2012-2013 season. In the 2011-2012 season, 8481 unique non-up-to-date children had visits. Slightly more non-up-to-date children seen when the reminder was 'on' were vaccinated than when 'off' (76.2% vs 73.8%; P = .027). Effects were seen in the winter (67.9% vs 62.2%; P = .005), not fall (76.8% vs 76.5%). The reminder also increased documentation of the reason for vaccine non-administration (68.1% vs 41.5%; P < .0001). During the 2011-2012 season, the reminder displayed for 8630 unique visits, and clinicians interacted with it in 83.1% of cases where patients required vaccination. During the 2012-2013 season, it displayed for 22 248 unique visits; clinicians interacted with it in 84.8% of cases. An IIS-linked influenza vaccination reminder increased vaccination later in the winter when fewer vaccine doses are usually given. Although the reminder did not require clinicians to interact with it, they frequently did; utilization did not wane over time. Copyright © 2015 by the American Academy of Pediatrics.

Structural basis for glucose-6-phosphate activation of glycogen synthase

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

Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.

2010-11-22

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by themore » binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.« less

Acid-Sensitive Sheddable PEGylated PLGA Nanoparticles Increase the Delivery of TNF-α siRNA in Chronic Inflammation Sites

PubMed Central

Aldayel, Abdulaziz M; Naguib, Youssef W; O'Mary, Hannah L; Li, Xu; Niu, Mengmeng; Ruwona, Tinashe B; Cui, Zhengrong

2016-01-01

There has been growing interest in utilizing small interfering RNA (siRNA) specific to pro-inflammatory cytokines, such as tumor necrosis factor-α ( TNF-α), in chronic inflammation therapy. However, delivery systems that can increase the distribution of the siRNA in chronic inflammation sites after intravenous administration are needed. Herein we report that innovative functionalization of the surface of siRNA-incorporated poly (lactic-co-glycolic) acid (PLGA) nanoparticles significantly increases the delivery of the siRNA in the chronic inflammation sites in a mouse model. The TNF-α siRNA incorporated PLGA nanoparticles were prepared by the standard double emulsion method, but using stearoyl-hydrazone-polyethylene glycol 2000, a unique acid-sensitive surface active agent, as the emulsifying agent, which renders (i) the nanoparticles PEGylated and (ii) the PEGylation sheddable in low pH environment such as that in chronic inflammation sites. In a mouse model of lipopolysaccharide-induced chronic inflammation, the acid-sensitive sheddable PEGylated PLGA nanoparticles showed significantly higher accumulation or distribution in chronic inflammation sites than PLGA nanoparticles prepared with an acid-insensitive emulsifying agent (i.e., stearoyl-amide-polyethylene glycol 2000) and significantly increased the distribution of the TNF-α siRNA incorporated into the nanoparticles in inflamed mouse foot. PMID:27434685

Molecular insights of protein contour recognition with ligand pharmacophoric sites through combinatorial library design and MD simulation in validating HTLV-1 PR inhibitors.

PubMed

Selvaraj, Chandrabose; Omer, Ankur; Singh, Poonam; Singh, Sanjeev Kumar

2015-01-01

Retroviruses HIV-1 and HTLV-1 are chiefly considered to be the most dangerous pathogens in Homo sapiens. These two viruses have structurally unique protease (PR) enzymes, which are having common function of its replication mechanism. Though HIV PR drugs failed to inhibit HTLV-1 infections, they emphatically emphasise the need for designing new lead compounds against HTLV-1 PR. Therefore, we tried to understand the binding level interactions through the charge environment present in both ligand and protein active sites. The domino effect illustrates that libraries of purvalanol-A are attuned to fill allosteric binding site of HTLV-1 PR through molecular recognition and shows proper binding of ligand pharmacophoric features in receptor contours. Our screening evaluates seven compounds from purvalanol-A libraries, and these compounds' pharmacophore searches for an appropriate place in the binding site and it places well according to respective receptor contour surfaces. Thus our result provides a platform for the progress of more effective compounds, which are better in free energy calculation, molecular docking, ADME and molecular dynamics studies. Finally, this research provided novel chemical scaffolds for HTLV-1 drug discovery.

Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State*♦

PubMed Central

Fourati, Zaineb; Ruza, Reinis Reinholds; Laverty, Duncan; Drège, Emmanuelle; Delarue-Cochin, Sandrine; Joseph, Delphine; Koehl, Patrice; Smart, Trevor; Delarue, Marc

2017-01-01

Barbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations. Several derivatives of barbiturates containing anomalous scatterers were synthesized, and these derivatives helped us unambiguously identify a unique barbiturate binding site within the central ion channel pore in a closed conformation. In addition, docking calculations around the observed binding site for all three states of the receptor, including a model of the desensitized state, showed that barbiturates preferentially stabilize the closed state. The identification of this pore binding site sheds light on the mechanism of barbiturate inhibition of cationic pLGICs and allows the rationalization of several structural and functional features previously observed for barbiturates. PMID:27986812

Structural basis of arrestin-3 activation and signaling

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

Chen, Qiuyan; Perry, Nicole A.; Vishnivetskiy, Sergey A.

A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP6) is a non-receptor activator of arrestin-3 and report the structure of IP6-activated arrestin-3 at 2.4-Å resolution. IP6-activated arrestin-3 exhibits an inter-domain twist and a displaced C-tail, hallmarks of active arrestin. IP6 binds to the arrestin phosphate sensor, and is stabilized by trimerization. Analysis of the trimerization surface, which is also the receptor-binding surface, suggests a feature called the finger loop as a key region of the activation sensor. We show that finger loop helicity and flexibility may underliemore » coupling to hundreds of diverse receptors and also promote arrestin-3 activation by IP6. Importantly, we show that effector-binding sites on arrestins have distinct conformations in the basal and activated states, acting as switch regions. These switch regions may work with the inter-domain twist to initiate and direct arrestin-mediated signaling.« less

One Course, One Web Site--Of Course? Maybe Not!

ERIC Educational Resources Information Center

Cohn, Ellen R.

2004-01-01

Colleges and universities increasingly employ commercial Web-based course management systems (such as Blackboard and WebCT). How is it, then, that these institutions unquestioningly allocate a unique Web site to each class? Why establish one Web site for one course when other options provide so many benefits? Why isn't there a clamor for…

Examining the Presence of Social Media on University Web Sites

ERIC Educational Resources Information Center

Greenwood, Grant

2012-01-01

Over the past few years, social networking has exploded into a massive medium that has captured the attention of a large portion of the American population. The ever-growing social networking site(s) (SNS) movement has filled a networking gap and thus, has presented higher education institutions with unique opportunities (Reid 2009) to further…

Multifocal multi-site Warthin tumour.

PubMed

Hilton, Jennifer M; Phillips, John S; Hellquist, Henrik B; Premachandra, Don J

2008-12-01

The unique case of a 55-year-old man with multifocal adenolymphoma (Warthin's tumour) of both parotid glands, the neck and post-nasal space is presented. Warthin tumour is almost exclusively a parotid tumour but is known to be bilateral in 7-10% of cases and multifocal in 2% of cases. Most extraglandular Warthin tumours have been located in neck lymph nodes and only a few cases have been reported from other sites. The presented case is unique in having synchronous and metachronous Warthin tumours, as well as one of the tumours being neither truly parotid, nor within a lymph node.

Newberry EGS Seismic Velocity Model

DOE Data Explorer

Templeton, Dennise

2013-10-01

We use ambient noise correlation (ANC) to create a detailed image of the subsurface seismic velocity at the Newberry EGS site down to 5 km. We collected continuous data for the 22 stations in the Newberry network, together with 12 additional stations from the nearby CC, UO and UW networks. The data were instrument corrected, whitened and converted to single bit traces before cross correlation according to the methodology in Benson (2007). There are 231 unique paths connecting the 22 stations of the Newberry network. The additional networks extended that to 402 unique paths crossing beneath the Newberry site.

Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes

PubMed Central

Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B.

2016-01-01

Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5′→3′, 3′ →5′ or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically. Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm PMID:27515825

Single-Stranded Nucleic Acids Bind to the Tetramer Interface of SAMHD1 and Prevent Formation of the Catalytic Homotetramer.

PubMed

Seamon, Kyle J; Bumpus, Namandjé N; Stivers, James T

2016-11-08

Sterile alpha motif and HD domain protein 1 (SAMHD1) is a unique enzyme that plays important roles in nucleic acid metabolism, viral restriction, and the pathogenesis of autoimmune diseases and cancer. Although much attention has been focused on its dNTP triphosphohydrolase activity in viral restriction and disease, SAMHD1 also binds to single-stranded RNA and DNA. Here we utilize a UV cross-linking method using 5-bromodeoxyuridine-substituted oligonucleotides coupled with high-resolution mass spectrometry to identify the binding site for single-stranded nucleic acids (ssNAs) on SAMHD1. Mapping cross-linked amino acids on the surface of existing crystal structures demonstrated that the ssNA binding site lies largely along the dimer-dimer interface, sterically blocking the formation of the homotetramer required for dNTPase activity. Surprisingly, the disordered C-terminus of SAMHD1 (residues 583-626) was also implicated in ssNA binding. An interaction between this region and ssNA was confirmed in binding studies using the purified SAMHD1 583-626 peptide. Despite a recent report that SAMHD1 possesses polyribonucleotide phosphorylase activity, we did not detect any such activity in the presence of inorganic phosphate, indicating that nucleic acid binding is unrelated to this proposed activity. These data suggest an antagonistic regulatory mechanism in which the mutually exclusive oligomeric state requirements for ssNA binding and dNTP hydrolase activity modulate these two functions of SAMHD1 within the cell.

Design manual for low water stream crossings.

DOT National Transportation Integrated Search

1983-10-01

The purpose of this manual is to provide design guidelines for : low water stream crossings (LWSCs). Rigid criteria for determining the : applicability of a LWSC to a given site are not established since each : site is unique in terms of physical, so...