Science.gov

Sample records for active colony sites

  1. Measuring activity in ant colonies

    NASA Astrophysics Data System (ADS)

    Noda, C.; Fernández, J.; Pérez-Penichet, C.; Altshuler, E.

    2006-12-01

    Ants, as paradigm of social insects, have become a recurrent example of efficient problem solvers via self-organization. In spite of the simple behavior of each individual, the colony as a whole displays "swarm intelligence:" the organization of ant trails for foraging is a typical output of it. But conventional techniques of observation can hardly record the amount of data needed to get a detailed understanding of self-organization of ant swarms in the wild. Here we are presenting a measurement system intended to monitor ant activity in the field comprising massive data acquisition and high sensitivity. A central role is played by an infrared sensor devised specifically to monitor relevant parameters to the activity of ants through the exits of the nest, although other sensors detecting temperature and luminosity are added to the system. We study the characteristics of the activity sensor and its performance in the field. Finally, we present massive data measured at one exit of a nest of Atta insularis, an ant endemic to Cuba, to illustrate the potential of our system.

  2. Colonie Interim Storage Site annual site environmental report for calendar year 1989, Colonie, New York

    SciTech Connect

    Not Available

    1990-05-01

    IN 1984, Congress assigned the cleanup of the National Lead (NL) Industries site in Colonie, New York, to the Department of Energy (DOE) as part of a decontamination research and development project under the 1984 Energy and Water Appropriations Act. DOE then included the site in the Formerly Utilized Sites Remedial Action Program (FUSRAP), an existing DOE program to decontaminate or otherwise control sites where residual radioactive materials remain for the early years of the nation's atomic energy program. DOE instituted an environmental monitoring program at the site in 1984. Results are presented annually in reports such as this. Under FUSRAP, the first environmental monitoring report for this site presented data for calendar year 1984. This report presents the findings of the environmental monitoring program conducted during calendar year 1989. 16 refs., 17 figs., 14 tabs.

  3. Colony site dynamics and habitat use in Atlantic coast seabirds

    USGS Publications Warehouse

    Erwin, R.M.; Galli, J.; Burger, J.

    1981-01-01

    Seabird colony sizes and movements were documented in the DelMarVa coastal region in 1976-1977 and in New Jersey in 1978-1979. Most colonies were found on marsh and dredge deposition islands and on barrier island beaches. For the 'traditionally' beach-nesting Herring Gull, Common Tern, and Black Skimmer, larger, more stable colonies were found on barrier beaches than on marsh islands. In marsh habitats, rates of colony-site change of marshnesting Forster's Tern and Laughing Gulls were similar to those of the former beach nesters. Several adaptations have evolved in marsh specialists to cope with a high risk of reproductive failure due to flooding, but both Herring Gulls and Common Terns also appear to be very adaptable in nesting under various habitat conditions. New colonies and those abandoned between years may be pioneering attempts by younger or inexperienced birds, because they are often smaller than persistent colonies, although patterns differ among areas and habitats. Colony-site dynamics are complex and result from many selective factors including competition, predation, physical changes in site structure, and flooding. The invasion of Herring Gulls into marshes along the mid-Atlantic coast has had an impact on new colony-site choice by associated seabirds. Calculating colony-site turnover rates allows for comparisons among species, habitats, and regions and may give useful insights into habitat quality and change and alternative nesting strategies

  4. Nest site and colony characteristics of wading birds in selected atlantic coast colonies

    USGS Publications Warehouse

    Beaver, D.L.; Osborn, R.G.; Custer, T.W.

    1980-01-01

    Nests of 5 spp. of wading birds [Egretta thula, Plegadis falcinellus, Florida caerulea, casmerodius albus and Hydranassa tricolor] were identified and marked during the breeding season at 6 locations from Maccachusetts [USA] to North Carolina [USA]. At the end of the breeding season, 12 characteristics of nest-site location were measured. Nest locations were mapped to examine dispersion and nearest neighbor relationships. Multivariate analysis were used to describe and compare sites and species. Variations in nest-sites between colonies were greater than between species; colonies differed mainly in the variety and size of vegetation. Birds preferred to nest in vegetation that offered relatively stable nest sites, and the dispersion of nests in the colonies was related to vegetative patterns. The interaction of these factors with the number of bird species and the abundance of birds in the colony determined whether nest sites were stratified, segregated or randomly distributed.

  5. Distribution species abundance and nesting site use of Atlantic coast colonies of herons and their allies

    USGS Publications Warehouse

    Custer, T.W.; Osborn, R.G.; Stout, W.F.

    1980-01-01

    In 1975 and 1976, 8 teams of investigators located 262 colonies of nesting herons and their allies along the Atlantic coast from Florida to Maine [USA]. Fourteen species [Ajaia ajaja, Plegadis falcinellus, Nycticorax nycticorax, Ardea herodias, Eudocimus albus, Egretta thula, Hydranassa tricolor, Bubulcus ibis, Casmerodius albus, Butorides striatus, Florida caerulea, Dichromanassa rufescens, Nyctanassa violacea and Mycteria americana] were found in Florida, numbers decreasing to 7 in Maine. Colonies censused in the extreme south and north of the study area were lower in number of species and number of adults than those in the intermediate area. More than 90% of the colony sites surveyed in 1975 were active in 1976. The total number of nesting adults per colony, number of species per colony and number of nesting adults of each species per colony in 1976 were significantly correlated with their respective values for 1975. Abandoned and new colonies may be satellites of nearby reused colonies; they had fewer individuals and species than reused colonies and were closer to reused colonies than reused colonies were to each other. [This study was part of an attempt to examine colonially nesting herons as biological indicators of environmental quality.

  6. Derivation of guidelines for uranium residual radioactive material in soil at the Colonie Site, Colonie, New York

    SciTech Connect

    Dunning, D.

    1996-05-01

    Residual radioactive material guidelines for uranium in soil were derived for the Colonie site located in Colonie, New York. This site has been designated for remedial action under the Formerly Utilized Sites Remedial Action Program (FUSRAP) of the U.S. Department of Energy (DOE). The site became contaminated with radioactive material as a result of operations conducted by National Lead (NL) Industries from 1958 to 1984; these activities included brass foundry operations, electroplating of metal products, machining of various components using depleted uranium, and limited work with small amounts of enriched uranium and thorium. The Colonie site comprises the former NL Industries property, now designated the Colonie Interim Storage Site (CISS), and 56 vicinity properties contaminated by fallout from airborne emissions; 53 of the vicinity properties were previously remediated between 1984 and 1988. In 1984, DOE accepted ownership of the CISS property from NL Industries. Residual radioactive material guidelines for individual radionuclides and total uranium were derived on the basis of the requirement that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works in the immediate vicinity of the site should not exceed a dose of 30 mrem/yr following remedial action for the current use and likely future use scenarios or a dose of 100 mrem/yr for less likely future use scenarios. The DOE residual radioactive material guideline computer code, RESRAD, was used in this evaluation; RESRAD implements the methodology described in the DOE manual for establishing residual radioactive material guidelines.

  7. Distribution, species abundance, and nesting-site use of Atlantic coast colonies of herons and their allies

    USGS Publications Warehouse

    Custer, T.W.; Osborn, R.G.; Stout, W.F.

    1980-01-01

    In 1975 and 1976, eight teams of investigators located 262 colonies of nesting herons and their allies along the Atlantic coast from Florida to Maine. Fourteen species were found in Florida, numbers decreasing to seven in Maine. Colonies censused in the extreme south and north of the study area were lower in number of species and number of adults than those in the intermediate area. More than 90% of the colony sites surveyed in 1975 were active in 1976. The total number of nesting adults per colony, number of species per colony, and number of nestinga dults of each speciesp er colonyi n 1976 were significantlyc orrelatedw ith their respective values for 1975. Abandoned and new colonies appeared to be satellites of nearby reused colonies; they had fewer individuals and species than reused colonies and were closer to reused colonies than reused colonies were to each other.

  8. Work plan for the remedial investigation/feasibility study-environmental assessment for the Colonie site, Colonie, New York

    SciTech Connect

    Not Available

    1990-06-01

    This work plan has been prepared to document the scoping and planning process performed by the US Department of Energy (DOE) to support remedial action activities at the Colonie site. The site is located in eastern New York State in the town of Colonie near the city of Albany. Remedial action of the Colonie site is being planned as part of DOE's Formerly Utilized Sites Remedial Action Program. The DOE is responsible for controlling the release of all radioactive and chemical contaminants from the site. Under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), a remedial investigation/feasibility study (RI/FS) must be prepared to support the decision-making process for evaluating remedial action alternatives. This work plan contains a summary of information known about the site as of January 1988, presents a conceptual site model that identifies potential routes of human exposure to site containments, identifies data gaps, and summarizes the process and proposed studies that will be used to fill the data gaps. In addition, DOE activities must be conducted in compliance with the National Environmental Policy Act (NEPA), which requires consideration of the environmental consequences of a proposed action as part of its decision-making process. This work also describes the approach that will be used to evaluate potential remedial action alternatives and includes a description of the organization, project controls, and task schedules that will be employed to fulfill the requirements of both CERCLA and NEPA. 48 refs., 18 figs., 25 tabs.

  9. Ant colonies prefer infected over uninfected nest sites.

    PubMed

    Pontieri, Luigi; Vojvodic, Svjetlana; Graham, Riley; Pedersen, Jes Søe; Linksvayer, Timothy A

    2014-01-01

    During colony relocation, the selection of a new nest involves exploration and assessment of potential sites followed by colony movement on the basis of a collective decision making process. Hygiene and pathogen load of the potential nest sites are factors worker scouts might evaluate, given the high risk of epidemics in group-living animals. Choosing nest sites free of pathogens is hypothesized to be highly efficient in invasive ants as each of their introduced populations is often an open network of nests exchanging individuals (unicolonial) with frequent relocation into new nest sites and low genetic diversity, likely making these species particularly vulnerable to parasites and diseases. We investigated the nest site preference of the invasive pharaoh ant, Monomorium pharaonis, through binary choice tests between three nest types: nests containing dead nestmates overgrown with sporulating mycelium of the entomopathogenic fungus Metarhizium brunneum (infected nests), nests containing nestmates killed by freezing (uninfected nests), and empty nests. In contrast to the expectation pharaoh ant colonies preferentially (84%) moved into the infected nest when presented with the choice of an infected and an uninfected nest. The ants had an intermediate preference for empty nests. Pharaoh ants display an overall preference for infected nests during colony relocation. While we cannot rule out that the ants are actually manipulated by the pathogen, we propose that this preference might be an adaptive strategy by the host to "immunize" the colony against future exposure to the same pathogenic fungus. PMID:25372856

  10. Ant Colonies Prefer Infected over Uninfected Nest Sites

    PubMed Central

    Pontieri, Luigi; Vojvodic, Svjetlana; Graham, Riley; Pedersen, Jes Søe; Linksvayer, Timothy A.

    2014-01-01

    During colony relocation, the selection of a new nest involves exploration and assessment of potential sites followed by colony movement on the basis of a collective decision making process. Hygiene and pathogen load of the potential nest sites are factors worker scouts might evaluate, given the high risk of epidemics in group-living animals. Choosing nest sites free of pathogens is hypothesized to be highly efficient in invasive ants as each of their introduced populations is often an open network of nests exchanging individuals (unicolonial) with frequent relocation into new nest sites and low genetic diversity, likely making these species particularly vulnerable to parasites and diseases. We investigated the nest site preference of the invasive pharaoh ant, Monomorium pharaonis, through binary choice tests between three nest types: nests containing dead nestmates overgrown with sporulating mycelium of the entomopathogenic fungus Metarhizium brunneum (infected nests), nests containing nestmates killed by freezing (uninfected nests), and empty nests. In contrast to the expectation pharaoh ant colonies preferentially (84%) moved into the infected nest when presented with the choice of an infected and an uninfected nest. The ants had an intermediate preference for empty nests. Pharaoh ants display an overall preference for infected nests during colony relocation. While we cannot rule out that the ants are actually manipulated by the pathogen, we propose that this preference might be an adaptive strategy by the host to “immunize” the colony against future exposure to the same pathogenic fungus. PMID:25372856

  11. Colony Rheology: Active Arthropods Generate Flows

    NASA Astrophysics Data System (ADS)

    Daniels, Karen; Mann, Michael; Charbonneau, Patrick

    2015-03-01

    Hydrodynamic-like flows are observed in biological systems as varied as bacteria, insects, birds, fish, and mammals. Both the phenomenology (e.g. front instabilities, milling motions) and the interaction types (hydrodynamic, direct contact, psychological, excluded-volume) strongly vary between systems, but a question common to all of them is to understand the role of particle-scale fluctuations in controlling large-scale rheological behaviors. We will address these questions through experiments on a new system, Tyrolichus casei (cheese mites), which live in dense, self-mixing colonies composed of a mixture of living mites and inert flour/detritus. In experiments performed in a Hele-Shaw geometry, we observe that the rheology of a colony is strongly dependent on the relative concentration of active and inactive particles. In addition to spreading flows, we also observe that the system can generate convective circulation and auto-compaction.

  12. Colony density and activity times of the ant Camponotus semitestaceus (Hymenoptera: formicidae) in a shrub steppe community

    SciTech Connect

    Gano, K.A.; Rogers, L.E.

    1983-11-01

    Colony densities and above-ground activity periods were determined for Camponotus semitestaceus colonies within a shrub-steppe community. Colony densities (anti-x +/- SD) averaged 0.088 +/- 0.032 per m/sup 2/ and 0.048 +/- 0.028 per m/sup 2/ on two sagebrush-bunchgrass sites an

  13. Colonial America.

    ERIC Educational Resources Information Center

    Web Feet K-8, 2001

    2001-01-01

    Presents resources for grades K-8, on the subject of Colonial America. Describes Web sites; CD-ROMs and software; videos; books; audios; magazines; and professional resources. Includes two articles, "Native Americans in the Colonies," and "The Golden Age of Pirates," which also highlight resources. Presents a Web activity focusing on daily life in…

  14. Colony site selection and abandonment by least terns Sterna antillarum in New Jersey, USA

    USGS Publications Warehouse

    Kotliar, Natasha B.; Burger, Joanna

    1986-01-01

    The presence of shells or pebbles in a sandy substrate, and short, sparse vegetation, were the habitat characteristics of New Jersey least tern colony sites most strongly correlated with colony site selection. Dredge spoil sites had significantly greater evidence of human disturbance, distance to water, and proportion of coarse particles in the substrate than beach sites. These differences may have contributed to the smaller colonines and greater colony turnover rates at spoil sites relative to beach sites. Overall, abandoned colony site characteristics did not differ significantly from occupied sites. However, human disturbance, over-growth of vegetation, predation, and flooding were all prevalent at colonies prior to abandonment. The results of this study suggest techniques for habitat management of both least and little terns.

  15. Vocal activity as a low cost and scalable index of seabird colony size.

    PubMed

    Borker, Abraham L; McKown, Matthew W; Ackerman, Joshua T; Eagles-Smith, Collin A; Tershy, Bernie R; Croll, Donald A

    2014-08-01

    Although wildlife conservation actions have increased globally in number and complexity, the lack of scalable, cost-effective monitoring methods limits adaptive management and the evaluation of conservation efficacy. Automated sensors and computer-aided analyses provide a scalable and increasingly cost-effective tool for conservation monitoring. A key assumption of automated acoustic monitoring of birds is that measures of acoustic activity at colony sites are correlated with the relative abundance of nesting birds. We tested this assumption for nesting Forster's terns (Sterna forsteri) in San Francisco Bay for 2 breeding seasons. Sensors recorded ambient sound at 7 colonies that had 15-111 nests in 2009 and 2010. Colonies were spaced at least 250 m apart and ranged from 36 to 2,571 m(2) . We used spectrogram cross-correlation to automate the detection of tern calls from recordings. We calculated mean seasonal call rate and compared it with mean active nest count at each colony. Acoustic activity explained 71% of the variation in nest abundance between breeding sites and 88% of the change in colony size between years. These results validate a primary assumption of acoustic indices; that is, for terns, acoustic activity is correlated to relative abundance, a fundamental step toward designing rigorous and scalable acoustic monitoring programs to measure the effectiveness of conservation actions for colonial birds and other acoustically active wildlife. PMID:24628442

  16. Vocal activity as a low cost and scalable index of seabird colony size

    USGS Publications Warehouse

    Borker, Abraham L.; McKown, Matthew W.; Ackerman, Joshua T.; Eagles-Smith, Collin A.; Tershy, Bernie R.; Croll, Donald A.

    2014-01-01

    Although wildlife conservation actions have increased globally in number and complexity, the lack of scalable, cost-effective monitoring methods limits adaptive management and the evaluation of conservation efficacy. Automated sensors and computer-aided analyses provide a scalable and increasingly cost-effective tool for conservation monitoring. A key assumption of automated acoustic monitoring of birds is that measures of acoustic activity at colony sites are correlated with the relative abundance of nesting birds. We tested this assumption for nesting Forster's terns (Sterna forsteri) in San Francisco Bay for 2 breeding seasons. Sensors recorded ambient sound at 7 colonies that had 15–111 nests in 2009 and 2010. Colonies were spaced at least 250 m apart and ranged from 36 to 2,571 m2. We used spectrogram cross-correlation to automate the detection of tern calls from recordings. We calculated mean seasonal call rate and compared it with mean active nest count at each colony. Acoustic activity explained 71% of the variation in nest abundance between breeding sites and 88% of the change in colony size between years. These results validate a primary assumption of acoustic indices; that is, for terns, acoustic activity is correlated to relative abundance, a fundamental step toward designing rigorous and scalable acoustic monitoring programs to measure the effectiveness of conservation actions for colonial birds and other acoustically active wildlife.

  17. Colonie Interim Storage Site: Annual site environmental report, Colonie, New York, Calendar year 1986: Formerly Utilized Sites Remedial Action Program (FUSRAP)

    SciTech Connect

    Not Available

    1987-06-01

    During 1986, the environmental monitoring program continued at the Colonie Interim Storage Site (CISS), a US Department of Energy (DOE) facility located in Colonie, New York. The CISS is part of the Formerly Utilized Sites Remedial Action Program (FUSRAP), a DOE program to decontaminate or otherwise control sites where residual radioactive materials remain from the early years of the nation's atomic energy program or from commercial operations causing conditions that Congress has mandated DOE to remedy. As part of the decontamination research and development project authorized by Congress under the 1984 Energy and Water Appropriations Act, remedial action is being conducted at the site and at vicinity properties by Bechtel National Inc. (BNI), Project Management Contractor for FUSRAP. The environmental monitoring program is also carried out by BNI. The monitoring program at the CISS measures external gamma radiation levels as well as uranium and radium-226 concentrations in surface water, groundwater, and sediment. To verify that the site is in compliance with the DOE radiation protection standard and to assess the potential effect of the site on public health, the radiation dose was calculated for the maximally exposed individual. Based on the conservative scenario described in the report, the maximally exposed individual would receive an annual external exposure approximately equivalent to 5% of the DOE radiation protection standard of 100 mrem/y. Results of 1986 monitoring show that the CISS is in compliance with the DOE radiation protection standard. 14 refs., 9 figs., 9 tabs.

  18. Colonie Interim Storage Site environmental report for calendar year 1992, 1130 Central Avenue, Colonie, New York. Formerly Utilized Sites Remedial Action Program (FUSRAP)

    SciTech Connect

    Not Available

    1993-05-01

    This report describes the environmental surveillance program at the Colonie Interim Storage Site (CISS) and provides the results for 1992. The site is located in eastern New York State, approximately 6.4 km (4.0 mi) northwest of downtown Albany. From 1958 to 1984, National Lead (NL) Industries used the facility to manufacture various components from depleted and enriched uranium natural thorium. Environmental monitoring of CISS began in 1984 when Congress added, the site to the US Department of Energy`s (DOE) Formerly Utilized Sites Remedial Action Program (FUSRAP). FUSRAP is a program established to identify and decontaminate or otherwise control sites where residual radioactive materials remain from the early years of the nation`s atomic energy program or from commercial operations causing conditions that Congress has authorized DOE to remedy. The environmental surveillance program at CISS includes sampling networks for external gamma radiation exposure and for thorium-232 and total uranium concentrations in surface water, sediment, and groundwater. Several chemical parameters are also measured in groundwater, including total metals, volatile organics, and water quality parameters. This surveillance program assists in fulfilling the DOE policy of measuring and monitoring effluents from DOE activities and calculating hypothetical doses. Results are compared with applicable Environmental Protection Agency (EPA) and New York State Department of Environmental Conservation (NYSDEC) standards, DOE derived concentration guides (DCGs), dose limits, and other DOE requirements.

  19. Development of Preliminary Remediation Goals for Indoor Dust at the Colonie FUSRAP Site - 12273

    SciTech Connect

    Watters, David J.; Opdyke, Clifford P.; Moore, James T.

    2012-07-01

    The Colonie FUSRAP Site is located in the Town of Colonie, Albany County, New York. The U.S. Army Corps of Engineers is currently addressing environmental contamination associated with the Site under the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) process as part of the Formerly Utilized Sites Remedial Action Program (FUSRAP). Soil remediation activities have been substantially completed at the Colonie FUSRAP Site and its vicinity properties under the FUSRAP. A study unrelated to FUSRAP was recently performed by an independent party to establish the distribution of DU contamination in various media in the environs of the Site. As part of this study, dust samples were collected in residencies and businesses in the immediate vicinity of the Site. These samples were collected in non-living areas such as basement window sills and garages. Many of these samples tested positive for DU. An assessment was performed to establish preliminary remediation goals (PRGs) for indoor dust in non-living areas of residential homes and businesses in the vicinity of the Site. The results of this assessment provide estimates of dose-based, carcinogenic risk-based, and noncarcinogenic-based PRGs derived from a hypothetical exposure scenario with reasonable levels of conservatism. Ultimately, the PRGs will be compared to results of dust sampling and analyses in residences and businesses in proximity of the Site to determine whether a response action is appropriate. This assessment estimates PRGs for DU contaminated dust in non-living areas of residences in the vicinity of the Colonie FUSRAP site based on a reasonably conservative exposure scenario. Estimated PRGs based on residential receptors are considered to be conservatively representative of workers in nearby businesses based on the considerably longer exposure duration of residents relative to workers. This assessment provides reasonably conservative estimates of PRGs for DU contaminated dust in non

  20. Active mechanics and geometry of adherent cells and cell colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2014-03-01

    Measurements of traction stresses exerted by adherent cells or cell colonies on elastic substrates have yielded new insight on how the mechanical and geometrical properties of the substrate regulate cellular force distribution, mechanical energy, spreading, morphology or stress ber architecture. We have developed a generic mechanical model of adherent cells as an active contractile gel mechanically coupled to an elastic substrate and to neighboring cells in a tissue. The contractile gel model accurately predicts the distribution of cellular and traction stresses as observed in single cell experiments, and captures the dependence of cell shape, traction stresses and stress ber polarization on the substrate's mechanical and geometrical properties. The model further predicts that the total strain energy of an adherent cell is solely regulated by its spread area, in agreement with recent experiments on micropatterned substrates with controlled geometry. When used to describe the behavior of colonies of adherent epithelial cells, the model demonstrates the crucial role of the mechanical cross-talk between intercellular and extracellular adhesion in regulating traction force distribution. Strong intercellular mechanical coupling organizes traction forces to the colony periphery, whereas weaker intercellular coupling leads to the build up of traction stresses at intercellular junctions. Furthermore, in agreement with experiments on large cohesive keratinocyte colonies, the model predicts a linear scaling of traction forces with the colony size. This scaling suggests the emergence of an effective surface tension as a scale-free material property of the adherent tissue, originating from actomyosin contractility.

  1. Colonie Interim Storage Site environmental surveillance report for calendar year 1993

    SciTech Connect

    Not Available

    1994-06-01

    This report summarizes the results of environmental surveillance activities conducted at the Colonie Interim Storage Site (CISS) during calendar year 1993. It includes an overview of site operations, the basis for radiological and nonradiological monitoring, dose to the offsite population, and summaries of environmental programs at CISS. Environmental surveillance activities were conducted in accordance with the site environmental monitoring plan, which describes the rationale and design criteria for the surveillance program, the frequency of sampling and analysis, specific sampling and analysis procedures, and quality assurance requirements. Appendix A contains a discussion of the nature of radiation, the way it is measured, and common sources of it. The primary environmental guidelines and limits applicable to CISS are given in US Department of Energy (DOE) orders and mandated by six federal acts: the Clean Air Act; the Clean Water Act; the Resource Conservation and Recovery Act (RCRA); the Toxic Substances Control Act; the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA); and the National Environmental Policy Act (NEPA). DOE began environmental monitoring of CISS in 1984 when DOE was authorized by Congress through the Energy and Water Development Appropriations Act to conduct a decontamination research and development program at the site. The site was subsequently assigned to DOE`s Formerly Utilized Sites Remedial Action Program (FUSRAP).

  2. Random mitotic activities across human embryonic stem cell colonies.

    SciTech Connect

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L.

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  3. The use of decoys to attract Least Terns (Sterna antillarum) to abandoned colony sites in New Jersey

    USGS Publications Warehouse

    Kotliar, Natasha B.; Burger, Joanna

    1984-01-01

    The number of Least Tern colony sites in New Jersey has declined in recent years. Decoys were used at two recently abandoned Least Tern colony sites in New Jersey to encourage nesting. The sites were chosen because of their apparent suitability as colony sites and the relative ease of protecting them from human disturbance and predators. Least Terns were observed flying over and landing at both sites, although nesting occurred at only one site. The effect of decoys was statically significant at the colony site used for nesting. At this site, 44.5% of the landings occurred in the plot containing decoys and only 10.6% o the landings were in the control plot. Nesting was initiated among the decoys. These results indicate that decoys can be used to attract Least Terns to abandoned colony sites and may be useful for managing Least Terns and other colonial nesting birds.

  4. Collective chemotaxis and segregation of active bacterial colonies.

    PubMed

    Ben Amar, M

    2016-01-01

    Still recently, bacterial fluid suspensions have motivated a lot of works, both experimental and theoretical, with the objective to understand their collective dynamics from universal and simple rules. Since some species are active, most of these works concern the strong interactions that these bacteria exert on a forced flow leading to instabilities, chaos and turbulence. Here, we investigate the self-organization of expanding bacterial colonies under chemotaxis, proliferation and eventually active-reaction. We propose a simple model to understand and quantify the physical properties of these living organisms which either give cohesion or on the contrary dispersion to the colony. Taking into account the diffusion and capture of morphogens complicates the model since it induces a bacterial density gradient coupled to bacterial density fluctuations and dynamics. Nevertheless under some specific conditions, it is possible to investigate the pattern formation as a usual viscous fingering instability. This explains the similarity and differences of patterns according to the physical bacterial suspension properties and explain the factors which favor compactness or branching. PMID:26888040

  5. Collective chemotaxis and segregation of active bacterial colonies

    PubMed Central

    Amar, M. Ben

    2016-01-01

    Still recently, bacterial fluid suspensions have motivated a lot of works, both experimental and theoretical, with the objective to understand their collective dynamics from universal and simple rules. Since some species are active, most of these works concern the strong interactions that these bacteria exert on a forced flow leading to instabilities, chaos and turbulence. Here, we investigate the self-organization of expanding bacterial colonies under chemotaxis, proliferation and eventually active-reaction. We propose a simple model to understand and quantify the physical properties of these living organisms which either give cohesion or on the contrary dispersion to the colony. Taking into account the diffusion and capture of morphogens complicates the model since it induces a bacterial density gradient coupled to bacterial density fluctuations and dynamics. Nevertheless under some specific conditions, it is possible to investigate the pattern formation as a usual viscous fingering instability. This explains the similarity and differences of patterns according to the physical bacterial suspension properties and explain the factors which favor compactness or branching. PMID:26888040

  6. Landscape changes and colony site dynamics: How gull-billed terns cope at the sea's edge

    USGS Publications Warehouse

    Erwin, R.M.; Williams, B.; Watts, B.; Truitt, B.; Stotts, D.; Eyler, B.

    1996-01-01

    Gull-billed Terns have declined dramatically in coastal Virginia over the past 20 years, with apparently low reproductive success. They nest, usually in mixed-species colonies, in two discrete habitat types: large, sandy barrier islands or shell/sandbars on the edges of marsh islands in the lagoon systems. The smaller shell/sandbars seem to provide more consistent nestling habitat and predation pressures than do barrier islands among years. We hypothesize that colony site turnover (between years) should be higher in the more uncertain barrier island habitats than among the shell/sandbar colonies. Our results do not corroborate the prediction. We postulate that social (and other) factors may explain these differences.

  7. Modeling colony site dynamics: a case study of gull-billed terns (Sterna nilotica) in coastal Virginia

    USGS Publications Warehouse

    Erwin, R.M.; Nichols, J.D.; Eyler, T.B.; Stotts, D.B.; Truitt, B.R.

    1998-01-01

    We developed a Markov process model for colony site dynamics of Gull-billed Terns (Sterna nilotica) in coastal Virginia. We used the model and data on colony site occupation from 1993 to 1996 to estimate model parameters. Each year, we monitored the breeding numbers of Gull-billed Terns and their frequent colony associates, Common Terns (Sterna hirundo) and Black Skimmers (Rynchops niger) at colony sites along about 80 km of the barrier island region of Virginia. We also monitored flooding events and renesting. We developed the model for colony survival, extinction, and recolonization at potential colony sites over the four-year period. We then used data on annual site occupation by Gull-billed Terns to estimate model parameters and test between different structures reflecting competing hypotheses. Results revealed a dynamic system, but provided no evidence that the dynamics were Markovian , i.e. the probability of occupancy of a site in one year was not influenced by whether it had been occupied the previous year. Nor did the colony-level reproductive success the previous season seem to affect the probability of site occupancy. Site survival and recolonization rates were similar, and the overall annual probability of a site being occupied over the course of the four-year period was estimated to be 0.59 Of the total of 25 sites that were used during the four-year period, 16 were used in only one or two years while only three were used all four years.. Flooding and renesting were frequent in both habitat types in all years. The frequent flooding of nests on shellpiles argues for more effective management; augmentation with shell and sand to increase elevations as little as 20 cm could have reduced flooding at a number of sites. The low colony-site fidelity we demonstrate suggests that an effective management approach is to provide a large number of alternative sand and/or shellpile sites that the terns may use. Sites not used one year may still be used in subsequent

  8. Harvester Ant Colony Variation in Foraging Activity and Response to Humidity

    PubMed Central

    Gordon, Deborah M.; Dektar, Katherine N.; Pinter-Wollman, Noa

    2013-01-01

    Collective behavior is produced by interactions among individuals. Differences among groups in individual response to interactions can lead to ecologically important variation among groups in collective behavior. Here we examine variation among colonies in the foraging behavior of the harvester ant, Pogonomyrmex barbatus. Previous work shows how colonies regulate foraging in response to food availability and desiccation costs: the rate at which outgoing foragers leave the nest depends on the rate at which foragers return with food. To examine how colonies vary in response to humidity and in foraging rate, we performed field experiments that manipulated forager return rate in 94 trials with 17 colonies over 3 years. We found that the effect of returning foragers on the rate of outgoing foragers increases with humidity. There are consistent differences among colonies in foraging activity that persist from year to year. PMID:23717415

  9. Colony density and activity times of the ant Camponotus semitestaceus (hymenoptera: Formicidae) in a shrub steppe community

    SciTech Connect

    Gano, K.A.; Rogers, L.E.

    1983-11-01

    Colony densities and above-ground activity periods were determined for Camponotus semitestaceus colonies within a shrub-steppe community. Colony densities (anti x=/- SD) averaged 0.088 +/- 0.032 per m/sup 2/ and 0.048 +/- 0.028 per m/sup 2/ on two sagebrush-bunchgrass sites and 0.028 +/- 0.028 per m/sup 2/ on a burned site. Seventy-five percent of the nest entrances were located alongside the stems of sagebrush, indicating a preference for these microhabitats as nest locations. Above-ground activity times were determined by using time lapse photography. Activity commenced shortly after sunset, when light intensities dropped to 2.5 to 1.0 foot-candles (ca. 27 to 11 lux) and terminated just before sunrise. Light intensity appears to be the primary cue for controlling above-ground activity periods of this species, but temperature also appears to be an important factor. When soil surface temperatures drop to 1.7 to 3.9/sup 0/C, all above-ground activity ceases, irrespective of light intensity.

  10. Colony density and activity times of the ant Camponotus semitestaceus (hymenoptera:formicidae) in a shrub steppe community

    SciTech Connect

    Gano, K.A.; Rogers, L.E.

    1983-11-01

    Colony densities and above-ground activity periods were determined for Camponotus semitestaceus colonies within a shrub-steppe community. Colony densities (x +/- SD) averaged 0.088 +/- 0.032 per m/sup 2/ and 0.048 +/- 0.028 per m/sup 2/ on two sagebrush-bunchgrass sites and 0.028 +/- 0.028 per m/sup 2/ on a burned site. Seventy-five percent of the nest entrances were located alongside the stems of sagebrush, indicating a preference for these microhabitats as nest locations. Above-ground activity times were determined by using time lapse photography. Activity commenced shortly after sunset, when light intensities dropped to 2.5 to 1.0 foot-candles (ca. 27 to 11 lux) and terminated just before sunrise. Light intensity appears to be the primary cue for controlling above-ground activity periods of this species, but temperature also appears to be an important factor. When soil surface temperatures drop to 1.7 to 3.9/sup 0/C, all above-ground activity ceases, irrespective of light intensity. 19 references, 3 figures, 2 tables.

  11. A binary ant colony optimization classifier for molecular activities.

    PubMed

    Hammann, Felix; Suenderhauf, Claudia; Huwyler, Jörg

    2011-10-24

    Chemical fingerprints encode the presence or absence of molecular features and are available in many large databases. Using a variation of the Ant Colony Optimization (ACO) paradigm, we describe a binary classifier based on feature selection from fingerprints. We discuss the algorithm and possible cross-validation procedures. As a real-world example, we use our algorithm to analyze a Plasmodium falciparum inhibition assay and contrast its performance with other machine learning paradigms in use today (decision tree induction, random forests, support vector machines, artificial neural networks). Our algorithm matches established paradigms in predictive power, yet supplies the medicinal chemist and basic researcher with easily interpretable results. Furthermore, models generated with our paradigm are easy to implement and can complement virtual screenings by additionally exploiting the precalculated fingerprint information. PMID:21854036

  12. Granulocyte colony-stimulating factor improves alternative activation of microglia under microenvironment of spinal cord injury.

    PubMed

    Guo, Y; Zhang, H; Yang, J; Liu, S; Bing, L; Gao, J; Hao, A

    2013-05-15

    Granulocyte colony-stimulating factor (G-CSF) was investigated in the present study to examine whether it could affect the activation status of microglia under microenvironment of spinal cord injury and provide a potential therapeutic treatment for spinal cord injury. We established mouse spinal cord hemisection model and injected recombinant human G-CSF (rhG-CSF) subcutaneously. The results demonstrated that G-CSF could recruit microglia to the injury site in the first 72h after spinal cord injury. Moreover, G-CSF inhibits the expression of pro-inflammatory factors and promotes the expression of neurotrophic factors. Additionally, G-CSF also increases the expression of markers of M2 macrophage and inhibits the expression of markers of M1 macrophage in BV2 microglia in vitro model, favoring the M2 polarization of microglia under the microenvironment of spinal cord hemisection. NFκB signal pathway was involved in G-CSF-induced polarization of BV2 microglia. As a conclusion, we suggested that administration of G-CSF within the first 72h after spinal cord injury might reduce early inflammation-induced detrimental effect and promote an anti-inflammatory response that favors repair via improving alternative activation of microglia. Administration of G-CSF in the acute phase of spinal cord injury may be a promising strategy in restorative therapy after spinal cord injury. PMID:23419550

  13. Breeding colonies of least terns (Sternula antillarum) in northern Sonora, Mexico, 2006-2008

    USGS Publications Warehouse

    Rosemartin, Alyssa; van Riper, Charles, III

    2012-01-01

    We document distribution of breeding least terns (Sternula antillarum) in northern Sonora, Mexico, 2006-2008. We report breeding activity at six sites with active colonies, including three previously undocumented colonies.

  14. [Chang Sei Kim's activities on public health in colonial Korea].

    PubMed

    Park, Yunjae

    2006-12-01

    in Korea was rejected by Rockefeller Foundation, as the policy of foundation was to finance only government institutes, not private ones. Resigning his position at Severance Medical College in 1927. Chang Sei Kim went to Shanghai to work as a Field Director in the Council on Health Education. The council was affiliated with the Rockefeller Foundation and was founded to ameliorate the hygienic situation in China. He was well fitted to the job, because China, like Korea, shared the aim to achieve independence by promoting better health for its people and because he could be appointed as a public officer which could not happen in colonial Korea. To solve the ever-serious problems with tuberculosis in China, he went again to the U. S. to conduct research and raise money for the establishment of a sanitarium. Chang Sei Kim passed away there in 1934 at the age of 42. PMID:17575705

  15. The patterns of seasonal activity of Ixodes vespertilionis (Acari: Ixodidae) on Rhinolophus hipposideros in nursery colonies.

    PubMed

    Piksa, Krzysztof; Górz, Andrzej; Nowak-Chmura, Magdalena; Siuda, Krzysztof

    2014-02-01

    The aim of this study was to describe the dynamics of the long-legged bat tick Ixodes vespertilionis infestation on the lesser horseshoe bat Rhinolophus hipposideros in 2 nursery colonies roosting in attics. Out of a total of 810 lesser horseshoe bats examined, 217 (26.8%) were found to be infested with a total of 464 I. vespertilionis individuals. The developmental stage most frequently found was the larva, followed by the nymph, and the adult female. Bats were significantly more frequently infested with I. vespertilionis ticks in the period April to May than in other months. In these months, all tick developmental stages were observed. During summer and autumn, only immature developmental stages were recorded, whilst in September and October larvae predominated. Considerable differences in tick load between nursery colonies were observed. The length of seasonal presence on bats, prevalence, and infestation intensity of I. vespertilionis on lesser horseshoe bats were higher in the nursery colony situated in close vicinity of a cave than in the colony situated far from the caves. The results suggest that the pattern of seasonal infestation of ticks on bats roosting in nursery colonies coincides with the seasonal activity of Rh. hipposideros in the caves. The first case of mixed infestation of the lesser horseshoe bat with I. vespertilionis and I. ricinus were also recorded. PMID:24252260

  16. Foraging Habitat Quality Constrains Effectiveness of Artificial Nest-Site Provisioning in Reversing Population Declines in a Colonial Cavity Nester

    PubMed Central

    Catry, Inês; Franco, Aldina M. A.; Rocha, Pedro; Alcazar, Rita; Reis, Susana; Cordeiro, Ana; Ventim, Rita; Teodósio, Joaquim; Moreira, Francisco

    2013-01-01

    Among birds, breeding numbers are mainly limited by two resources of major importance: food supply and nest-site availability. Here, we investigated how differences in land-use and nest-site availability affected the foraging behaviour, breeding success and population trends of the colonial cavity-dependent lesser kestrel Falco naumanni inhabiting two protected areas. Both areas were provided with artificial nests to increase nest-site availability. The first area is a pseudo-steppe characterized by traditional extensive cereal cultivation, whereas the second area is a previous agricultural zone now abandoned or replaced by forested areas. In both areas, lesser kestrels selected extensive agricultural habitats, such as fallows and cereal fields, and avoided scrubland and forests. In the second area, tracked birds from one colony travelled significantly farther distances (6.2 km ±1.7 vs. 1.8 km ±0.4 and 1.9 km ±0.6) and had significant larger foraging-ranges (144 km2 vs. 18.8 and 14.8 km2) when compared to the birds of two colonies in the extensive agricultural area. Longer foraging trips were reflected in lower chick feeding rates, lower fledging success and reduced chick fitness. Availability and occupation of artificial nests was high in both areas but population followed opposite trends, with a positive increment recorded exclusively in the first area with a large proportion of agricultural areas. Progressive habitat loss around the studied colony in the second area (suitable habitat decreased from 32% in 1990 to only 7% in 2002) is likely the main driver of the recorded population decline and suggests that the effectiveness of bird species conservation based on nest-site provisioning is highly constrained by habitat quality in the surrounding areas. Therefore, the conservation of cavity-dependent species may be enhanced firstly by finding the best areas of remaining habitat and secondly by increasing the carrying capacity of high-quality habitat areas

  17. Induction of megakaryocytic colony-stimulating activity in mouse skin by inflammatory agents and tumor promoters

    SciTech Connect

    Clark, D.A.; Dessypris, E.N.; Koury, M.J.

    1987-03-01

    The production of megakaryocytic colony-stimulating activity (MEG-CSA) was assayed in acetic acid extracts of skin from mice topically treated with inflammatory and tumor-promoting agents. A rapid induction of MEG-CSA was found in skin treated both with phorbol 12-myristate 13-acetate (PMA), a strong tumor promoter, and with mezerein, a weak tumor promoter, but no induction was found in untreated skin. The time course of induction of MEG-CSA following treatment of skin with PMA or mezerein was very similar to that previously demonstrated for the induction of granulocyte-macrophage colony-stimulating activity in mouse skin by these agents. The induced MEG-CSA was found in both the epidermis and the dermis. Pretreatment of the skin with US -methasone abrogated the MEG-CSA induction. The cell number response curve suggests that the MEG-CSA acts directly on the progenitor cells of the megakaryocyte colonies. That topical administration of diterpene esters results in the rapid, local induction of MEG-CSA which can be blocked by US -methasone pretreatment suggests a mechanism for the thrombocytosis associated with some inflammatory states. The indirect action in which diterpene esters induce in certain cells the production or release of growth regulatory factors for other cell types may also aid in understanding their carcinogenic properties.

  18. Influence of site-specific geology on oil shale fragmentation experiments at the Colony Mine, Garfield County, Colorado

    SciTech Connect

    Ray, J.M.; Harper, M.D.; Craig, J.L.; Edwards, C.L.

    1982-01-01

    The Los Alamos National Laboratory executed 19 intermediate scale cratering experiments in oil shale at the Colony Mine in Garfield County, Colorado. These experiments have led to a better understanding of fracture characteristics and fragmentation of in situ oil shale by use of a conventional high explosive. Geologic site characterization included detailed mapping, coring, and sample analyses. Site-specific geology was observed to be a major influence on the resulting crater geometry. The joint patterns at the experimental site frequently defined the final crater symmetry. Secondary influences included vugs, lithology changes, and grade fluctuations in the local stratigraphy. Most experiments, in both the rib and floor, were conducted to obtain data to investigate the fragmentation results within the craters. The rubble was screened for fragment-size distributions. Geologic features in proximity to the explosive charge had minimal effect on the rubble due to the overpowering effect of the detonation. However, these same features became more influential on the fracture and rubble characteristics with greater distances from the shothole. Postshot cores revealed a direct relationship between the grade of the oil shale and its susceptibility to fracturing. The Colony Mine experiments have demonstrated the significant role of geology in high explosive/oil shale interaction. It is probable that this role will have to be considered for larger applications to blast patterns and potential problems in retort stability in the future of oil shale development.

  19. A discrete artificial bee colony algorithm for detecting transcription factor binding sites in DNA sequences.

    PubMed

    Karaboga, D; Aslan, S

    2016-01-01

    The great majority of biological sequences share significant similarity with other sequences as a result of evolutionary processes, and identifying these sequence similarities is one of the most challenging problems in bioinformatics. In this paper, we present a discrete artificial bee colony (ABC) algorithm, which is inspired by the intelligent foraging behavior of real honey bees, for the detection of highly conserved residue patterns or motifs within sequences. Experimental studies on three different data sets showed that the proposed discrete model, by adhering to the fundamental scheme of the ABC algorithm, produced competitive or better results than other metaheuristic motif discovery techniques. PMID:27173272

  20. Salt site performance assessment activities

    SciTech Connect

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

    1983-01-01

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

  1. Characterization of the Active Microbiotas Associated with Honey Bees Reveals Healthier and Broader Communities when Colonies are Genetically Diverse

    PubMed Central

    Mattila, Heather R.; Rios, Daniela; Walker-Sperling, Victoria E.; Roeselers, Guus; Newton, Irene L. G.

    2012-01-01

    Recent losses of honey bee colonies have led to increased interest in the microbial communities that are associated with these important pollinators. A critical function that bacteria perform for their honey bee hosts, but one that is poorly understood, is the transformation of worker-collected pollen into bee bread, a nutritious food product that can be stored for long periods in colonies. We used 16S rRNA pyrosequencing to comprehensively characterize in genetically diverse and genetically uniform colonies the active bacterial communities that are found on honey bees, in their digestive tracts, and in bee bread. This method provided insights that have not been revealed by past studies into the content and benefits of honey bee-associated microbial communities. Colony microbiotas differed substantially between sampling environments and were dominated by several anaerobic bacterial genera never before associated with honey bees, but renowned for their use by humans to ferment food. Colonies with genetically diverse populations of workers, a result of the highly promiscuous mating behavior of queens, benefited from greater microbial diversity, reduced pathogen loads, and increased abundance of putatively helpful bacteria, particularly species from the potentially probiotic genus Bifidobacterium. Across all colonies, Bifidobacterium activity was negatively correlated with the activity of genera that include pathogenic microbes; this relationship suggests a possible target for understanding whether microbes provide protective benefits to honey bees. Within-colony diversity shapes microbiotas associated with honey bees in ways that may have important repercussions for colony function and health. Our findings illuminate the importance of honey bee-bacteria symbioses and examine their intersection with nutrition, pathogen load, and genetic diversity, factors that are considered key to understanding honey bee decline. PMID:22427917

  2. Characterization of the active microbiotas associated with honey bees reveals healthier and broader communities when colonies are genetically diverse.

    PubMed

    Mattila, Heather R; Rios, Daniela; Walker-Sperling, Victoria E; Roeselers, Guus; Newton, Irene L G

    2012-01-01

    Recent losses of honey bee colonies have led to increased interest in the microbial communities that are associated with these important pollinators. A critical function that bacteria perform for their honey bee hosts, but one that is poorly understood, is the transformation of worker-collected pollen into bee bread, a nutritious food product that can be stored for long periods in colonies. We used 16S rRNA pyrosequencing to comprehensively characterize in genetically diverse and genetically uniform colonies the active bacterial communities that are found on honey bees, in their digestive tracts, and in bee bread. This method provided insights that have not been revealed by past studies into the content and benefits of honey bee-associated microbial communities. Colony microbiotas differed substantially between sampling environments and were dominated by several anaerobic bacterial genera never before associated with honey bees, but renowned for their use by humans to ferment food. Colonies with genetically diverse populations of workers, a result of the highly promiscuous mating behavior of queens, benefited from greater microbial diversity, reduced pathogen loads, and increased abundance of putatively helpful bacteria, particularly species from the potentially probiotic genus Bifidobacterium. Across all colonies, Bifidobacterium activity was negatively correlated with the activity of genera that include pathogenic microbes; this relationship suggests a possible target for understanding whether microbes provide protective benefits to honey bees. Within-colony diversity shapes microbiotas associated with honey bees in ways that may have important repercussions for colony function and health. Our findings illuminate the importance of honey bee-bacteria symbioses and examine their intersection with nutrition, pathogen load, and genetic diversity, factors that are considered key to understanding honey bee decline. PMID:22427917

  3. Flight activity of 4-lb Australian package bee colonies used for almond pollination.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing acreage of almonds in California has increased the demand for honey bee colonies for pollination. Since 2005, domestic U.S. colonies have been supplemented with colonies started from package bees imported from Australia. The need for almond pollination in late winter in California fits we...

  4. Acaricidal activity of Swietenia mahogani and Swietenia macrophylla ethanolic extracts against Varroa destructor in honeybee colonies.

    PubMed

    El Zalabani, Soheir M; El-Askary, Hesham I; Mousa, Ola M; Issa, Marwa Y; Zaitoun, Ahmed A; Abdel-Sattar, Essam

    2012-02-01

    The acaricidal (miticidal) activity of 90% ethanolic extracts of leaves and stem bark of Swietenia mahogani and Swietenia macrophylla were tested against Varroa destructor mite. Four concentrations were used over two different time intervals under laboratory and field conditions. In general, it was noticed that the acaricidal effect based on mortality and LC(50) of all tested extracts against the Varroa mite was concentration and time dependant. The acaricidal action against Varroa mites was relatively the least for the S. macrophylla stem bark extract at 500 ppm concentration after 48 h while it reached 100% and 95% in case of S. mahogani bark and S. macrophylla leaves, respectively. The% infestation with Varroa in colonies treated with the different extracts at various time intervals showed that the rate of infestation decreased to 0.0% after 12 days from the beginning of treatments with 500 ppm of S. mahogani leaves extract compared to 0.79% decrease after treatment with Mitac, a reference drug (60 mg/colony). The rate of infestation in case of treatments with S. mahogani bark, S. macrophylla leaves and S. macrophylla bark was decreased to 0.11%, 2.41% and 1.08%, respectively. The highest reduction was observed with S. mahogani leaves extract followed by S. mahogani bark. All the tested extracts showed less or no effect on honey bees at the different concentrations and at different bioassay times. This study suggested that the use of natural plant extracts or their products as ecofriendly biodegradable agents could be of high value for the control of Varroa mite. PMID:22101075

  5. Musical Acculturation through Primary School Activities during Japanese Colonial Rule of Korea (1910-1945)

    ERIC Educational Resources Information Center

    Kim, Jeong Ha

    2014-01-01

    Global colonialism and continuing post-colonial influences caused widespread cultural change at the interface of different cultures. Musical acculturation can be observed in most colonised countries. Some pro-colonialists apologetically allege that through colonisation the colonised territories would receive developmental aid and economical…

  6. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  7. Wading birds as biological indicators 1975 colony survey

    USGS Publications Warehouse

    Custer, T.W.; Osborn, R.G.

    1977-01-01

    The suitability of wading birds (herons and their allies) as biological indicators in the coastal environment were studied in 1975 by 8 teams of investigators which located and censused 198 colonies along the Atlantic coast from Maine to Florida [USA]. Over 1/4 million breeding birds [Ardea herodias, Butorides virescens, Florida caerulea, Bubulcus ibis, Dichromanassa rufescens, Casmerodius albus, Egretta thula, Hydranassa tricolor, Nycticorax nycticorax, Nyctanassa violacea, Mycteria americana, Plegadis falcinellus, Eudocimus albus and Ajaia ajaja] were censused. The number of species in colonies ranged from 1-11. The number of 1- and 2-spp. colonies increased from Florida to Maine. Colony size decreased from Florida to Maine. Wading bird colony sites are generally active each year and the number of colonies may have recently increased in some areas of the coast. Species composition and total population of colonies fluctuate from year to year. The breeding population of wading birds was correlated with the area of coastal wetlands by state. Five teams of investigators studied the reproductive biology of 9 spp. in 13 colonies. Mean clutch size, the percentage of nests in which 1 or more eggs hatched and the overall percentage of eggs that hatched differed among colonies for some species, but no latitudinal gradient was found in any of these characteristics for any species. The use of wading birds to their full potential as biological indicators requires further exploration: survey and reproductive success methods need to be tested, the survey of colonies repeated, available historical information assembled and habitat requirements measured.

  8. Front structure and dynamics in dense colonies of motile bacteria: Role of active turbulence.

    PubMed

    Chatterjee, Rayan; Joshi, Abhijeet A; Perlekar, Prasad

    2016-08-01

    We study the spreading of a bacterial colony undergoing turbulentlike collective motion. We present two minimalistic models to investigate the interplay between population growth and coherent structures arising from turbulence. Using direct numerical simulation of the proposed models we find that turbulence has two prominent effects on the spatial growth of the colony: (a) the front speed is enhanced, and (b) the front gets crumpled. Both these effects, which we highlight by using statistical tools, are markedly different in our two models. We also show that the crumpled front structure and the passive scalar fronts in random flows are related in certain regimes. PMID:27627334

  9. Front structure and dynamics in dense colonies of motile bacteria: Role of active turbulence

    NASA Astrophysics Data System (ADS)

    Chatterjee, Rayan; Joshi, Abhijeet A.; Perlekar, Prasad

    2016-08-01

    We study the spreading of a bacterial colony undergoing turbulentlike collective motion. We present two minimalistic models to investigate the interplay between population growth and coherent structures arising from turbulence. Using direct numerical simulation of the proposed models we find that turbulence has two prominent effects on the spatial growth of the colony: (a) the front speed is enhanced, and (b) the front gets crumpled. Both these effects, which we highlight by using statistical tools, are markedly different in our two models. We also show that the crumpled front structure and the passive scalar fronts in random flows are related in certain regimes.

  10. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

    Finding quality Internet resources for high school students is a continuing challenge. Several high-quality web sites are presented for educators and students. These sites offer activities to learn how an art conservator looks at paintings, create a newspaper, research and develop an end product, build geometry and physics skills, explore science…

  11. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

    PubMed Central

    Herter, Susanne; Kranz, David C; Turner, Nicholas J

    2015-01-01

    Summary Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

  12. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  13. Neuroprotective Activities of Granulocyte-Macrophage Colony Stimulating Factor Following Controlled Cortical Impact

    PubMed Central

    Kelso, Matthew L.; Elliott, Bret R.; Haverland, Nicole A.; Mosley, R. Lee; Gendelman, Howard E.

    2014-01-01

    Neurodegeneration after traumatic brain injury (TBI) is facilitated by innate and adaptive immunity and can be harnessed to effect brain repair. In mice subjected to controlled cortical impact (CCI) we show that treatment with granulocyte macrophage colony stimulating factor (GM-CSF) affects regulatory T cell numbers coincident with decreased lesion volumes and increased cortical tissue sparing. This paralleled increases in neurofilament and diminished reactive microglial staining. Transcriptomic analysis showed that GM-CSF induces robust immune neuroprotective responses seven days following CCI. Together, these results support the therapeutic potential of GM-CSF for TBI. PMID:25468272

  14. Comparison of within hive sampling and seasonal activity of Nosema ceranae in honey bee colonies.

    PubMed

    Traver, Brenna E; Williams, Matthew R; Fell, Richard D

    2012-02-01

    Nosema ceranae is a microsporidian parasite of the European honey bee, Apis mellifera, that is found worldwide and in multiple Apis spp.; however, little is known about the effects of N. ceranae on A. mellifera. Previous studies using spore counts suggest that there is no longer a seasonal cycle for N. ceranae and that it is found year round with little variation in infection intensity among months. Our goal was to determine whether infection levels differ in bees collected from different areas of the hive and if there may be seasonal differences in N. ceranae infections. A multiplex species-specific real-time PCR assay was used for the detection and quantification of N. ceranae. Colonies were sampled monthly from September 2009-2010 by collecting workers from honey supers, the fringe of the brood nest, and the brood nest. We found that all bees sampled were infected with N. ceranae and that there was no significant difference in infection levels among the different groups of bees sampled (P=0.74). However, significant differences in colony infection levels were found at different times of the year (P<0.01) with the highest levels in April-June and lower levels in the fall and winter. While our study was only performed for one year, it sheds light on the fact that there may be a seasonality to N. ceranae infections. Being able to predict future N. ceranae infections can be used to better advise beekeepers on N. ceranae management. PMID:22085836

  15. Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene.

    PubMed Central

    Fraser, J K; Guerra, J J; Nguyen, C Y; Indes, J E; Gasson, J C; Nimer, S D

    1994-01-01

    Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein. Images PMID:8114751

  16. Colony stimulating factor-inducing activity of isoflavone C-glucosides from the bark of Dalbergia monetaria.

    PubMed

    Kawaguchi, K; Alves, S de M; Watanabe, T; Kikuchi, S; Satake, M; Kumazawa, Y

    1998-10-01

    To obtain immunomodulating substances from Amazonian medicinal plants, hot water extracts from 21 samples available commercially were tested in terms of mitogenic and colony-stimulating factor (CSF)-inducing activities. Among them, Dalbergia monetaria exhibited the highest CSF-inducing activity. Orobol 8-C-glucoside (OCG-8) and orobol 6-C-glucoside (OCG-6) were isolated from the bark of D. monetaria as major constituents. The CSF-inducing activity of OCG-8 was higher than that of OCG-6 and a dose-dependent manner at a range of 0.1-10 mg/mouse. Serum CSF production induced by an intraperitoneal (i.p.) injection with 1 mg OCG-8 reached a peak at 4-6 h later, suggesting that OCG-8 would act on hematopoietic system. PMID:9810271

  17. Active site specificity of plasmepsin II.

    PubMed Central

    Westling, J.; Cipullo, P.; Hung, S. H.; Saft, H.; Dame, J. B.; Dunn, B. M.

    1999-01-01

    Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues. PMID:10548045

  18. Granulocyte-macrophage colony-stimulating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter.

    PubMed Central

    Sakamoto, K M; Fraser, J K; Lee, H J; Lehman, E; Gasson, J C

    1994-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event. Images PMID:8065330

  19. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

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

  20. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

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

  1. Robust boundary detection and tracking of left ventricles on ultrasound images using active shape model and ant colony optimization.

    PubMed

    Zhang, Yaonan; Gao, Yuan; Jiao, Jinling; Li, Xian; Li, Sai; Yang, Jun

    2014-01-01

    Information regarding the motion, strain and synchronization are important for cardiac diagnosis and therapy. Extraction of such information from ultrasound images remains an open problem till today. In this paper, a novel method is proposed to extract the boundaries of left ventricles and track these boundaries in ultrasound image sequences. The initial detection of boundaries was performed by an active shape model scheme. Subsequent refinement of the boundaries was done by using local variance information of the images. The main objective of this paper is the formulation of a new boundary tracking algorithm using ant colony optimization technique. The experiments conducted on the simulated image sequences and the real cardiac ultrasound image sequences shows a positive and promising result. PMID:25226995

  2. Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against Mycoplasma mycoides subspecies mycoides small colony type.

    PubMed

    Ayling, R D; Baker, S E; Nicholas, R A; Peek, M L; Simon, A J

    2000-02-26

    Minimum inhibitory concentrations (MIC) and minimum mycoplasmacidal concentrations (MMC) of the antimicrobials danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin were determined in vitro for 20 isolates of Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), the causative agent of contagious bovine pleuropneumonia (CBPP). The majority of strains were most susceptible to tilmicosin, followed by danofloxacin, oxytetracycline, florfenicol and spectinomycin with MIC50 values of 0.015, 0.25, 0.5, 1 and 8 microg/ml, and MMC50 values of 0.06, 0.5, 8, 8 and 16 microg/ml, respectively. However, tilmicosin had poor mycoplasmacidal activity against two recent strains from Portugal. There was no evidence of resistance to danofloxacin in any of the strains. PMID:10737293

  3. Inhibition of CD26 enzyme activity with pro-boropro stimulates rat granulocyte/macrophage colony formation and thymocyte proliferation in vitro.

    PubMed

    Bristol, L A; Bachovchin, W; Takács, L

    1995-06-15

    CD26 dipeptidyl peptidase (DPPIV) is involved in the regulation of proliferation of some hematopoietic and T-lineage cells. Here, we show that Pro-boropro a potent inhibitor of DPP activity has a costimulating effect in hematopoietic colony assays for macrophage and, to a lesser extent, for granulocyte colonies and has a stimulating effect in organ cultures of immature thymocytes. Based on these and other evidences, we propose that the mechanism by which CD26 regulates proliferation is associated with its DPP activity. PMID:7780144

  4. Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity.

    PubMed Central

    Gouilleux, F; Pallard, C; Dusanter-Fourt, I; Wakao, H; Haldosen, L A; Norstedt, G; Levy, D; Groner, B

    1995-01-01

    The molecular components which mediate cytokine signaling from the cell membrane to the nucleus were studied. Upon the interaction of cytokines with their receptors, members of the janus kinase (Jak) family of cytoplasmic protein tyrosine kinases and of the signal transducers and activators of transcription (Stat) family of transcription factors are activated through tyrosine phosphorylation. It has been suggested that the Stat proteins are substrates of the Jak protein tyrosine kinases. MGF-Stat5 is a member of the Stat family which has been found to confer the prolactin response. MGF-Stat5 can be phosphorylated and activated in its DNA binding activity by Jak2. The activation of MGF-Stat5 is not restricted to prolactin. Erythropoietin (EPO) and growth hormone (GH) stimulate the DNA binding activity of MGF-Stat5 in COS cells transfected with vectors encoding EPO receptor and MGF-Stat5 or vectors encoding GH receptor and MGF-Stat5. The activation of DNA binding by prolactin, EPO and GH requires the phosphorylation of tyrosine residue 694 of MGF-Stat5. The transcriptional induction of a beta-casein promoter luciferase construct in transiently transfected COS cells is specific for the prolactin activation of MGF-Stat5; it is not observed in EPO- and GH-treated cells. In the UT7 human hematopoietic cell line, EPO and granulocyte-macrophage colony stimulating factor activate the DNA binding activity of a factor closely related to MGF-Stat5 with respect to its immunological reactivity, DNA binding specificity and molecular weight. These results suggest that MGF-Stat5 regulates physiological processes in mammary epithelial cells, as well as in hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7744007

  5. Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells

    PubMed Central

    Kim, Seung Joon; Wan, Qiaoqiao; Cho, Eunhye; Han, Bumsoo; Yoder, Mervin C.; Voytik-Harbin, Sherry L.; Na, Sungsoo

    2014-01-01

    Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs. PMID:24393843

  6. Sailing to the Colonies.

    ERIC Educational Resources Information Center

    Carter, Dorothy S.

    1990-01-01

    Presents a class activity designed to foster an understanding of rules, develop analytical skills, and introduce students to early colonial history. Divides the class into groups who are sailing to the New World, and presents them with ethical and practical problems to be solved on board the ship. (RW)

  7. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  8. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  9. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1992-02-01

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

  10. Aerial surveys adjusted by ground surveys to estimate area occupied by black-tailed prairie dog colonies

    USGS Publications Warehouse

    Sidle, John G.; Augustine, David J.; Johnson, Douglas H.; Miller, Sterling D.; Cully, Jack F., Jr.; Reading, Richard P.

    2012-01-01

    Aerial surveys using line-intercept methods are one approach to estimate the extent of prairie dog colonies in a large geographic area. Although black-tailed prairie dogs (Cynomys ludovicianus) construct conspicuous mounds at burrow openings, aerial observers have difficulty discriminating between areas with burrows occupied by prairie dogs (colonies) versus areas of uninhabited burrows (uninhabited colony sites). Consequently, aerial line-intercept surveys may overestimate prairie dog colony extent unless adjusted by an on-the-ground inspection of a sample of intercepts. We compared aerial line-intercept surveys conducted over 2 National Grasslands in Colorado, USA, with independent ground-mapping of known black-tailed prairie dog colonies. Aerial line-intercepts adjusted by ground surveys using a single activity category adjustment overestimated colonies by ≥94% on the Comanche National Grassland and ≥58% on the Pawnee National Grassland. We present a ground-survey technique that involves 1) visiting on the ground a subset of aerial intercepts classified as occupied colonies plus a subset of intercepts classified as uninhabited colony sites, and 2) based on these ground observations, recording the proportion of each aerial intercept that intersects a colony and the proportion that intersects an uninhabited colony site. Where line-intercept techniques are applied to aerial surveys or remotely sensed imagery, this method can provide more accurate estimates of black-tailed prairie dog abundance and trends

  11. Granulocyte colony-stimulating factor does not enhance phagocytosis or microbicidal activity of human mature polymorphonuclear neutrophils in vitro.

    PubMed Central

    Shimono, N; Okada, K; Takeda, D; Eguchi, K; Misumi, H; Sawae, Y; Niho, Y

    1994-01-01

    The direct effects of human granulocyte colony-stimulating factor (hG-CSF) on mature polymorphonuclear neutrophils (PMNs) in vitro were studied with regard to chemotaxis, superoxide production, and phagocytosis and microbicidal activity against the following viable microorganisms: Staphylococcus aureus, serum-resistant Pseudomonas aeruginosa, and Candida albicans. Recombinant hG-CSF (rhG-CSF) acted as a chemoattractant for human PMNs in a dose-dependent manner. The chemotactic response of PMNs to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was not enhanced by rhG-CSF at any of the concentrations used. rhG-CSF did not induce the generation of superoxide by itself. However, rhG-CSF was able to prime human PMNs and to enhance O2- release stimulated by FMLP in a dose-dependent manner. rhg-CSF did not enhance phagocytosis or killing of the three species of microorganisms by normal PMNs. With PMNs obtained from patients who had hematological disorders or solid tumors, no enhancement of the microbicidal activity was observed in most cases. Microbial killing mediated by PMNs depended on the ratio of PMNs to target organisms. We concluded from these facts that the most important effect of rhG-CSF was to increase the number of the peripheral PMNs and not to enhance the functions of mature PMNs. PMID:8556501

  12. A Modified Active Appearance Model Based on an Adaptive Artificial Bee Colony

    PubMed Central

    Othman, Zulaiha Ali

    2014-01-01

    Active appearance model (AAM) is one of the most popular model-based approaches that have been extensively used to extract features by highly accurate modeling of human faces under various physical and environmental circumstances. However, in such active appearance model, fitting the model with original image is a challenging task. State of the art shows that optimization method is applicable to resolve this problem. However, another common problem is applying optimization. Hence, in this paper we propose an AAM based face recognition technique, which is capable of resolving the fitting problem of AAM by introducing a new adaptive ABC algorithm. The adaptation increases the efficiency of fitting as against the conventional ABC algorithm. We have used three datasets: CASIA dataset, property 2.5D face dataset, and UBIRIS v1 images dataset in our experiments. The results have revealed that the proposed face recognition technique has performed effectively, in terms of accuracy of face recognition. PMID:25165748

  13. Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

    PubMed Central

    Rausch, O; Marshall, C J

    1997-01-01

    The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK. PMID:9032244

  14. Granulocyte colony-stimulating factor reprograms bone marrow stromal cells to actively suppress B lymphopoiesis in mice

    PubMed Central

    Day, Ryan B.; Bhattacharya, Deepta; Nagasawa, Takashi

    2015-01-01

    The mechanisms that mediate the shift from lymphopoiesis to myelopoiesis in response to infectious stress are largely unknown. We show that treatment with granulocyte colony-stimulating factor (G-CSF), which is often induced during infection, results in marked suppression of B lymphopoiesis at multiple stages of B-cell development. Mesenchymal-lineage stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells and osteoblasts, constitutively support B lymphopoiesis through the production of multiple B trophic factors. G-CSF acting through a monocytic cell intermediate reprograms these stromal cells, altering their capacity to support B lymphopoiesis. G-CSF treatment is associated with an expansion of CAR cells and a shift toward osteogenic lineage commitment. It markedly suppresses the production of multiple B-cell trophic factors by CAR cells and osteoblasts, including CXCL12, kit ligand, interleukin-6, interleukin-7, and insulin-like growth factor-1. Targeting bone marrow stromal cells is one mechanism by which inflammatory cytokines such as G-CSF actively suppress lymphopoiesis. PMID:25814527

  15. A catalog of Louisiana's nesting seabird colonies

    USGS Publications Warehouse

    Fontenot, William R.; Cardiff, Steve W.; DeMay, Richard A.; Dittmann, Donna L.; Hartley, Stephen B.; Jeske, Clinton W.; Lorenz, Nicole; Michot, Thomas C.; Purrington, Robert Dan; Seymour, Michael; Vermillion, William G.

    2012-01-01

    collective habitats which comprise Louisiana's now fragile coastal zone have taken major hits from commercial/residential, oil & gas, and other industrial development, primarily in the form of coastal erosion exacerbated by these and other factors (Portnoy 1978, Spendelow and Patton 1988, Martin and Lester 1990, Green, et al. 2006). Moreover, during this same period, both geologic subsidence rates (Tornqvist et al. 2008) and mean sea-level (Tornqvist et al. 2002) have increased, along with significant tropical storm activity; all of which have combined to impact available marsh, barrier island, beach, and dredge spoil nesting habitat for waterbirds, especially seabirds, throughout the coastal zone of Louisiana. The primary objective of this publication is to detail those coastal Louisiana colonial seabird nesting sites for which we have reasonably accurate data, in a tabular, site-by-site format. All major survey (1976-2008) data of site-by-site seabird species counts, as well as several smaller data sets, referred to in the site history tables as “miscellaneous observations” obtained during the May-June seabird breeding period, are included. It is our hope that these data will provide a dependable foundation from which future colonial seabird nesting surveys might be planned and carried out, as well as showcase the importance of coastal Louisiana's seabird rookeries, and contribute to their conservation.

  16. Application of ant colony optimization in development of models for prediction of anti-HIV-1 activity of HEPT derivatives.

    PubMed

    Zare-Shahabadi, Vali; Abbasitabar, Fatemeh

    2010-09-01

    Quantitative structure-activity relationship models were derived for 107 analogs of 1-[(2-hydroxyethoxy) methyl]-6-(phenylthio)thymine, a potent inhibitor of the HIV-1 reverse transcriptase. The activities of these compounds were investigated by means of multiple linear regression (MLR) technique. An ant colony optimization algorithm, called Memorized_ACS, was applied for selecting relevant descriptors and detecting outliers. This algorithm uses an external memory based upon knowledge incorporation from previous iterations. At first, the memory is empty, and then it is filled by running several ACS algorithms. In this respect, after each ACS run, the elite ant is stored in the memory and the process is continued to fill the memory. Here, pheromone updating is performed by all elite ants collected in the memory; this results in improvements in both exploration and exploitation behaviors of the ACS algorithm. The memory is then made empty and is filled again by performing several ACS algorithms using updated pheromone trails. This process is repeated for several iterations. At the end, the memory contains several top solutions for the problem. Number of appearance of each descriptor in the external memory is a good criterion for its importance. Finally, prediction is performed by the elitist ant, and interpretation is carried out by considering the importance of each descriptor. The best MLR model has a training error of 0.47 log (1/EC(50)) units (R(2) = 0.90) and a prediction error of 0.76 log (1/EC(50)) units (R(2) = 0.88). PMID:20575016

  17. Granulocyte colony-stimulating factor impairs CD8(+) T cell functionality by interfering with central activation elements.

    PubMed

    Bunse, C E; Tischer, S; Lahrberg, J; Oelke, M; Figueiredo, C; Blasczyk, R; Eiz-Vesper, B

    2016-07-01

    Besides mobilizing stem cells into the periphery, granulocyte colony-stimulating factor (G-CSF) has been shown to influence various types of innate and adaptive immune cells. For example, it impairs the effector function of cytotoxic T lymphocytes (CTLs). It is assumed that this effect is mediated indirectly by monocytes, regulatory T cells and immunomodulatory cytokines influenced by G-CSF. In this study, isolated G-CSF-treated CD8(+) T cells were stimulated antigen-dependently with peptide-major histocompatibility complex (pMHC)-coupled artificial antigen-presenting cells (aAPCs) or stimulated antigen-independently with anti-CD3/CD28 stimulator beads. By measuring the changes in interferon (IFN)-γ and granzyme B expression at the mRNA and protein level, we showed for the first time that G-CSF has a direct effect on CD8(+) CTLs, which was confirmed based on the reduced production of IFN-γ and granzyme B by the cytotoxic T cell line TALL-104 after G-CSF treatment. By investigating further elements affected by G-CSF in CTLs from stem cell donors and untreated controls, we found a decreased phosphorylation of extracellular-regulated kinase (ERK)1/2, lymphocyte-specific protein tyrosine kinase (Lck) and CD3ζ after G-CSF treatment. Additionally, miRNA-155 and activation marker expression levels were reduced. In summary, our results show that G-CSF directly influences the effector function of cytotoxic CD8(+) T cells and affects various elements of T cell activation. PMID:26990855

  18. Biotinylated granulocyte/macrophage colony-stimulating factor analogues: effect of linkage chemistry on activity and binding.

    PubMed

    Angelotti, T P; Clarke, M F; Longino, M A; Emerson, S G

    1991-01-01

    Biotinylated granulocyte/macrophage colony-stimulating factor (GM-CSF) analogues with different linkage chemistries and levels of conjugated biotin were synthesized by reacting recombinant human GM-CSF with sulfosuccinimidyl 6-biotinamidohexanoate or biotin hydrazide/1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide. These chemically reactive forms of biotin produced derivatives biotinylated at amine or carboxyl groups, respectively. Amine-derivatized analogues of 1.2 and 3.8 mol of biotin/mol of protein (N1-bGM-CSF and N4-bGM-CSF) and a carboxyl-modified analogue of 4.6 mol of biotin/mol of protein (C5-bGM-CSF) were synthesized. These analogues were compared to determine the effect of biotinylation on biological activity and GM-CSF receptor binding characteristics. The biotinylated proteins migrated with the same molecular weight as the native, unmodified protein as determined by SDS-PAGE and could be detected by Western blotting with alkaline phosphatase conjugated streptavidin, thus demonstrating the biotin linkage. All three analogues retained full agonist activity relative to the native protein (EC50 = 10-15 pM) when assayed for the stimulation of human bone marrow progenitor cell growth. Cell surface GM-CSF receptor binding was characterized by the binding of the analogues to human neutrophils, with detection by fluorescein-conjugated avidin and fluorescence-activated cell sorting. The N-bGM-CSFs demonstrated GM-CSF receptor specific binding that was displaceable by excess underivatized protein, with the detected fluorescence signal decreasing with increasing biotin to protein molar ratio. In contrast, C5-bGM-CSF binding above background fluorescence could not be detected using this system, suggesting that this derivative could bind to and activate the receptor, but not simultaneously bind fluorescein-conjugated avidin. The amine-derivatized biotinylated GM-CSF analogues retained biological activity, could specifically label cell surface receptors, and may be

  19. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  20. A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells.

    PubMed

    Dias, Juliana Vieira; Benslimane-Ahmim, Zahia; Egot, Marion; Lokajczyk, Anna; Grelac, Françoise; Galy-Fauroux, Isabelle; Juliano, Luiz; Le-Bonniec, Bernard; Takiya, Cristina Maeda; Fischer, Anne-Marie; Blanc-Brude, Olivier; Morandi, Verônica; Boisson-Vidal, Catherine

    2012-10-15

    Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 μg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. PMID:22796565

  1. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  2. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  3. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  5. Substrate Binding and Active Site Residues in RNases E and G

    PubMed Central

    Garrey, Stephen M.; Blech, Michaela; Riffell, Jenna L.; Hankins, Janet S.; Stickney, Leigh M.; Diver, Melinda; Hsu, Ying-Han Roger; Kunanithy, Vitharani; Mackie, George A.

    2009-01-01

    The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5′-sensor, the S1 domain, and the Mg+2/Mn+2 binding sites. The effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the kcat decrease ∼40- to 100-fold) and RNA binding consistent with a key role for the 5′-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition. PMID:19778900

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

    SciTech Connect

    Hartman, F.C.

    1991-01-01

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

  7. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

    Background A common assumption about enzyme active sites is that their structures are highly conserved to specifically distinguish between closely similar compounds. However, with the discovery of distinct enzymes with similar reaction chemistries, more and more studies discussing the structural flexibility of the active site have been conducted. Results Most of the existing works on the flexibility of active sites focuses on a set of pre-selected active sites that were already known to be flexible. This study, on the other hand, proposes an analysis framework composed of a new data collecting strategy, a local structure alignment tool and several physicochemical measures derived from the alignments. The method proposed to identify flexible active sites is highly automated and robust so that more extensive studies will be feasible in the future. The experimental results show the proposed method is (a) consistent with previous works based on manually identified flexible active sites and (b) capable of identifying potentially new flexible active sites. Conclusions This proposed analysis framework and the former analyses on flexibility have their own advantages and disadvantage, depending on the cause of the flexibility. In this regard, this study proposes an alternative that complements previous studies and helps to construct a more comprehensive view of the flexibility of enzyme active sites. PMID:21342563

  8. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

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

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

    SciTech Connect

    Not Available

    1990-07-01

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

  10. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

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

  11. Bait distribution among multiple colonies of Pharaoh ants (hymenoptera: Formicidae).

    PubMed

    Oi, D H; Vail, K M; Williams, D F

    2000-08-01

    Pharaoh ant, Monomorium pharaonis (L.), infestations often consist of several colonies located at different nest sites. To achieve control, it is desirable to suppress or eliminate the populations of a majority of these colonies. We compared the trophallactic distribution and efficacy of two ant baits, with different modes of action, among groups of four colonies of Pharaoh ants. Baits contained either the metabolic-inhibiting active ingredient hydramethylnon or the insect growth regulator (IGR) pyriproxyfen. Within 3 wk, the hydramethylnon bait reduced worker and brood populations by at least 80%, and queen reductions ranged between 73 and 100%, when nests were in proximity (within 132 cm) to the bait source. However, these nest sites were reoccupied by ants from other colonies located further from the bait source. The pyriproxyfen bait was distributed more thoroughly to all nest locations with worker populations gradually declining by 73% at all nest sites after 8 wk. Average queen reductions ranged from 31 to 49% for all nest sites throughout the study. Even though some queens survived, brood reductions were rapid in the pyriproxyfen treatment, with reductions of 95% at all locations by week 3. Unlike the metabolic inhibitor, the IGR did not kill adult worker ants quickly, thus, more surviving worker ants were available to distribute the bait to all colonies located at different nest sites. Thus, from a single bait source, the slow-acting bait toxicant provided gradual, but long-term control, whereas the fast-acting bait toxicant provided rapid, localized control for a shorter duration. PMID:10985038

  12. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  13. Spleen volume varies with colony size and parasite load in a colonial bird.

    PubMed Central

    Brown, Charles R; Bomberger Brown, Mary

    2002-01-01

    Comparisons across bird species have indicated that those more exposed to parasites and pathogens invest more in immunological defence, as measured by spleen size. We investigated how spleen volume varied with colony size, parasite load and an individual's colony-size history in the cliff swallow, Petrochelidon pyrrhonota, a colonial passerine bird of North America. We used a sample of over 1700 birds that had all died during a period of inclement weather in 1996. We experimentally manipulated ectoparasitism by fumigating nests in some colonies prior to the bad weather. Birds from parasite-free colonies had significantly smaller spleens than those from naturally infested sites; spleen volume did not differ between the sexes and did not vary with age. Mean spleen volume increased significantly with the colony size at a site prior to the bad weather in 1996 and at the site in 1995, both measures of colony size being indices of ectoparasitism at a site. An individual's history of breeding-colony size (defined as the average colony size it had occupied in years prior to 1996) had no association with its spleen size. The results are consistent with parasite-induced splenomegaly whenever birds are exposed to large numbers of ectoparasites. The results do not support spleen size as being a signal of differential life-history investment in immunological defence among individuals and thus run counter to interpretations from recent cross-species comparisons. PMID:12079660

  14. Spleen volume varies with colony size and parasite load in a colonial bird.

    PubMed

    Brown, Charles R; Bomberger Brown, Mary

    2002-07-01

    Comparisons across bird species have indicated that those more exposed to parasites and pathogens invest more in immunological defence, as measured by spleen size. We investigated how spleen volume varied with colony size, parasite load and an individual's colony-size history in the cliff swallow, Petrochelidon pyrrhonota, a colonial passerine bird of North America. We used a sample of over 1700 birds that had all died during a period of inclement weather in 1996. We experimentally manipulated ectoparasitism by fumigating nests in some colonies prior to the bad weather. Birds from parasite-free colonies had significantly smaller spleens than those from naturally infested sites; spleen volume did not differ between the sexes and did not vary with age. Mean spleen volume increased significantly with the colony size at a site prior to the bad weather in 1996 and at the site in 1995, both measures of colony size being indices of ectoparasitism at a site. An individual's history of breeding-colony size (defined as the average colony size it had occupied in years prior to 1996) had no association with its spleen size. The results are consistent with parasite-induced splenomegaly whenever birds are exposed to large numbers of ectoparasites. The results do not support spleen size as being a signal of differential life-history investment in immunological defence among individuals and thus run counter to interpretations from recent cross-species comparisons. PMID:12079660

  15. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1997-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1995 through September 1996. The Radioactive Solid Waste Operations Group (RSWOG) of the Waste Management and Remedial Action Division (WMRAD) and the Environmental Sciences Division (ESD) at Oak Ridge National Laboratory (ORNL) established ASEMP in 1989. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North as required by Chapters 2 and 3 of US Department of Energy Order 5820.2A.

  16. Active sites environmental monitoring Program - Program Plan: Revision 2

    SciTech Connect

    Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

    1994-05-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results.

  17. The Amana Colonies.

    ERIC Educational Resources Information Center

    Lilja, Marilyn

    Designed for use in Iowa elementary schools, this unit introduces students to Iowa's Amana Colonies. Four lessons cover the history and cultural heritage of the colonies, daily life in historical times, daily life in modern times, and the colonies as a corporate museum. Throughout the lessons, emphasis is placed on the values and organization of…

  18. The effects of vitamin D binding protein-macrophage activating factor and colony-stimulating factor-1 on hematopoietic cells in normal and osteopetrotic rats.

    PubMed

    Benis, K A; Schneider, G B

    1996-10-15

    Osteopetrosis is a heterogeneous group of bone disorders characterized by the failure of osteoclasts to resorb bone and by several immunological defects including macrophage dysfunction. Two compounds, colony-stimulating factor-1 (CSF-1) and vitamin D-binding protein-macrophage activating factor (DBP-MAF) were used in the present study to evaluate their effects on the peritoneal population of cells and on cells within the bone marrow microenvironment in normal and incisors absent (ia) osteopetrotic rats. Previous studies in this laboratory have demonstrated that administration of DBP-MAF to newborn ia animals results in a substantial increase in bone marrow cavity size due to upregulated osteoclast function. To study the effects of these compounds on the macrophage/osteoclast precursors, DBP-MAF, CSF-1, and the combination of these compounds were given to newborn ia and normal littermate animals. Both the normal and mutant phenotypes responded similarly when treated with these compounds. Rats exhibited a profound shift toward the macrophage lineage from the neutrophil lineage when compared with vehicle-treated control animals after treatment with these compounds. In the in vivo peritoneal lavage study, animals received injections of CSF-1, DBP-MAF or DBP-MAF/CSF-1 over a 4-week period. The various types of cells in the peritoneal cavity were then enumerated. The in vitro study consisted of cells isolated from the bone marrow microenvironment and cultured on feeder layers of CSF-1, DBP-MAF, or DBP-MAF/CSF-1 for colony enumeration. The increase in macrophage numbers at the expense of neutrophil numbers could be seen in both the in vivo and in vitro experiments. The macrophage/osteoclast and neutrophil lineages have a common precursor, the granulocyte/macrophage colony-forming cell (GM-CFC). With the addition of CSF-1, the GM-CFC precursor may be induced into the macrophage/osteoclast lineage rather than the granulocyte lineage. This increased pool of cells in the

  19. Conceptual design of a lunar colony

    NASA Technical Reports Server (NTRS)

    Dalton, C. (Editor); Hohmann, E. (Editor)

    1972-01-01

    A systems engineering study is presented for a proposed lunar colony. The lunar colony was to grow from an existent, 12-man, earth-dependent lunar surface base and was to utilize lunar resources, becoming as earth-independent as possible. An in-depth treatment of some of the aspects of the lunar colony was given. We have found that the use of lunar resources is feasible for oxygen production (both for breathing and for space tug fuel), food production, and building materials. A program is outlined for recycling waste materials developed at the colony as well as a full program for growth and research activity of the colony to a level of 180 colonists. Recommendations for the lunar colony are given.

  20. The active site behaviour of electrochemically synthesised gold nanomaterials.

    PubMed

    Plowman, Blake J; O'Mullane, Anthony P; Bhargava, Suresh K

    2011-01-01

    Even though gold is the noblest of metals, a weak chemisorber and is regarded as being quite inert, it demonstrates significant electrocatalytic activity in its nanostructured form. It is demonstrated here that nanostructured and even evaporated thin films of gold are covered with active sites which are responsible for such activity. The identification of these sites is demonstrated with conventional electrochemical techniques such as cyclic voltammetry as well as a large amplitude Fourier transformed alternating current (FT-ac) method under acidic and alkaline conditions. The latter technique is beneficial in determining if an electrode process is either Faradaic or capacitive in nature. The observed behaviour is analogous to that observed for activated gold electrodes whose surfaces have been severely disrupted by cathodic polarisation in the hydrogen evolution region. It is shown that significant electrochemical oxidation responses occur at discrete potential values well below that for the formation of the compact monolayer oxide of bulk gold and are attributed to the facile oxidation of surface active sites. Several electrocatalytic reactions are explored in which the onset potential is determined by the presence of such sites on the surface. Significantly, the facile oxidation of active sites is used to drive the electroless deposition of metals such as platinum, palladium and silver from their aqueous salts on the surface of gold nanostructures. The resultant surface decoration of gold with secondary metal nanoparticles not only indicates regions on the surface which are rich in active sites but also provides a method to form interesting bimetallic surfaces. PMID:22455038

  1. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes. PMID:26990764

  2. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  3. Colonial America: A Course of Study.

    ERIC Educational Resources Information Center

    Bennett, Sondra; Stephens, Mark

    This illustrated unit of study can be incorporated into regular social studies courses in elementary classrooms. The unit focuses on life in the 13 original colonies from the settlement period to the Revolutionary War. Activities are provided to help students learn the names and locations of the colonies. A highlight of the unit is a study of the…

  4. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  5. Nest-size and colony characteristics of wading birds in selected Atlantic Coast colonies

    USGS Publications Warehouse

    Beaver, D.L.; Osborn, R.G.; Custer, T.W.

    1980-01-01

    Nests of 5 species of wading birds were identified and marked during the breeding season at 6 locations from Massachusetts to North Carolina. At the end of the breeding season 12 characteristics of nest-site location were measured. Nest locations were mapped to examine dispersion and nearest neighbor relationships. Multivariate analyses were used to describe and compare sites and species.....We found that variations in nest-sites between colonies were greater than between species; colonies differed mainly in the variety and size of vegetation; birds preferred to nest in vegetation that offered relatively stable nest-sites; and the dispersion of nests in the colonies was related to vegetative patterns. The interaction of these factors with the number of bird species and the abundance of birds in the colony seemed to determine whether nest-sites were stratified, segregated or randomly distributed.

  6. Site-specific effects on productivity of an upper trophic-level marine predator: Bottom-up, top-down, and mismatch effects on reproduction in a colonial seabird

    USGS Publications Warehouse

    Suryan, R.M.; Irons, D.B.; Brown, E.D.; Jodice, P.G.R.; Roby, D.D.

    2006-01-01

    We investigated the relative roles of bottom-up and top-down factors in limiting productivity of an upper trophic level marine predator. Our primary working hypothesis was that the reproductive success of black-legged kittiwakes (Rissa tridactyla) a piscivorous, colonial-nesting seabird, was most limited by the abundance, distribution, and species composition of surface-schooling forage fishes. A secondary working hypothesis was that reproductive loss to kittiwake nest predators was greatest during years of reduced prey availability. We report on a broad-scale, integrated study of kittiwakes and their prey in Prince William Sound, Alaska. Our study spanned five breeding seasons (1995-1999) and focused on three colonies that differed in size (ranging from ca. 220 to ca. 7000 breeding pairs) and proximity to each other (50-135 km apart). Kittiwakes in PWS encountered a variety of aquatic habitats, creating a complex foraging environment for breeding birds. We measured kittiwake reproductive success and foraging activities, while simultaneously measuring the abundance of surface schooling forage fishes throughout the foraging range of breeding kittiwakes. The abundance of primary prey species for kittiwakes (Pacific herring Clupea pallasi, Pacific sand lance Ammodytes hexapterus, and capelin Mallotus villosus) varied both annually and regionally, with no one region consistently having the greatest abundance of prey. Likewise, kittiwake reproductive success varied considerably among colonies and years. We found that bottom-up, top-down, timing mismatch, and colony-specific effects were all important to kittiwake productivity. Although bottom-up effects appeared to be strongest, they were not evident in some cases until other effects, such as geographic location (proximity of colony to prey concentrations) and top-down predation, were considered. Important bottom-up effects on kittiwake reproductive success were not only total prey abundance and distribution, but also

  7. Site-specific effects on productivity of an upper trophic-level marine predator: Bottom-up, top-down, and mismatch effects on reproduction in a colonial seabird

    NASA Astrophysics Data System (ADS)

    Suryan, Robert M.; Irons, David B.; Brown, Evelyn D.; Jodice, Patrick G. R.; Roby, Daniel D.

    2006-02-01

    We investigated the relative roles of bottom-up and top-down factors in limiting productivity of an upper trophic level marine predator. Our primary working hypothesis was that the reproductive success of black-legged kittiwakes ( Rissa tridactyla) a piscivorous, colonial-nesting seabird, was most limited by the abundance, distribution, and species composition of surface-schooling forage fishes. A secondary working hypothesis was that reproductive loss to kittiwake nest predators was greatest during years of reduced prey availability. We report on a broad-scale, integrated study of kittiwakes and their prey in Prince William Sound, Alaska. Our study spanned five breeding seasons (1995-1999) and focused on three colonies that differed in size (ranging from ca. 220 to ca. 7000 breeding pairs) and proximity to each other (50-135 km apart). Kittiwakes in PWS encountered a variety of aquatic habitats, creating a complex foraging environment for breeding birds. We measured kittiwake reproductive success and foraging activities, while simultaneously measuring the abundance of surface schooling forage fishes throughout the foraging range of breeding kittiwakes. The abundance of primary prey species for kittiwakes (Pacific herring Clupea pallasi, Pacific sand lance Ammodytes hexapterus, and capelin Mallotus villosus) varied both annually and regionally, with no one region consistently having the greatest abundance of prey. Likewise, kittiwake reproductive success varied considerably among colonies and years. We found that bottom-up, top-down, timing mismatch, and colony-specific effects were all important to kittiwake productivity. Although bottom-up effects appeared to be strongest, they were not evident in some cases until other effects, such as geographic location (proximity of colony to prey concentrations) and top-down predation, were considered. Important bottom-up effects on kittiwake reproductive success were not only total prey abundance and distribution, but also

  8. Colonial and post-colonial aspects of Australian identity.

    PubMed

    Tranter, Bruce; Donoghue, Jed

    2007-06-01

    Since the 1988 Bicentennial and the 2001 centenary of federation celebrations colonial images have flourished in Australia, highlighting the roles of convicts and free settlers during early colonization. Old sites, such as Port Arthur have been re-invigorated, and in 2004 Tasmanians celebrated the bicentenary of 'white' settlement. However, social scientists have given little attention to the role of colonial and post-colonial figures and myths as aspects of Australian national identity. We seek to address this issue by examining how convicts, free settlers, bushrangers and ANZACs are associated with contemporary identity in Australia. We examine evidence from the 2003 Australian Survey of Social Attitudes and find that historical figures such as the ANZACs and post-World War II immigrants comprise important aspects of national identity. A substantial majority of Australians judged ANZACs to be important, countering recent claims of the 'demise of the digger'. Sporting heroes are also at the core of Australian identity. Colonial figures appear to be far less important, although views on national identity vary according to social location. In particular, left-wing, university educated, younger, postmaterialist Australians view convicts and bushrangers as relatively important, indicating the salience of the larrikin in Australian identity. PMID:17610618

  9. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

    Andreeva, Julia; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciabà, Andrea; Tsaregorodtsev, Andrei

    2010-04-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  10. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  11. Thirty Years of Managing the Wreck of the Historic Australian Colonial-Built Schooner Clarence (1841-1850): From Ineffective to Pro-active Management

    NASA Astrophysics Data System (ADS)

    Harvey, Peter; Shefi, Debra

    2014-09-01

    Since its discovery in the early 1980s, the nineteenth-century Australian colonial-built schooner Clarence has undergone more extensive research and investigation than any other historic vessel located within Victoria's jurisdictional waters. Although early managerial approaches were well intentioned, those in-situ preservation methods employed proved ineffective and in some circumstances had an adverse effect, resulting in a significant loss of the archaeological integrity in a short 30-year period. As such, this paper outlines the knowledge gained due to both managerial successes and failures over the last 30 years, discusses the long-term applications of the applied methodologies and highlights the importance of environmental assessments, ongoing active management and the application of pro-active in-situ preservation methods.

  12. Thirty Years of Managing the Wreck of the Historic Australian Colonial-Built Schooner Clarence (1841-1850): From Ineffective to Pro-active Management

    NASA Astrophysics Data System (ADS)

    Harvey, Peter; Shefi, Debra

    2014-12-01

    Since its discovery in the early 1980s, the nineteenth-century Australian colonial-built schooner Clarence has undergone more extensive research and investigation than any other historic vessel located within Victoria's jurisdictional waters. Although early managerial approaches were well intentioned, those in-situ preservation methods employed proved ineffective and in some circumstances had an adverse effect, resulting in a significant loss of the archaeological integrity in a short 30-year period. As such, this paper outlines the knowledge gained due to both managerial successes and failures over the last 30 years, discusses the long-term applications of the applied methodologies and highlights the importance of environmental assessments, ongoing active management and the application of pro-active in-situ preservation methods.

  13. A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes

    SciTech Connect

    Roussel, M.F. ); Shurtleff, S.A.; Downing, J.R. ); Sherr, C.J. Saint Jude Children's Research Hospital, Memphis, TN )

    1990-09-01

    Substitution of phenylalanine for tyrosine-809 in the human colony-stimulating factor 1 receptor (CSF-1R) inhibited its ability to transduce ligand-dependent mitogenic signals in mouse NIH 3T3 cells. When combined with an activating mutation at codon 301 that induces constitutive CSF-1R tyrosine kinase activity, the codon 809 mutation suppressed ligand-independent cell transformation. Comparative mapping tryptic phosphopeptides from mutant and wild-type CSF-1R indicated that tyrosine-809 is a site of ligand-dependent receptor phosphorylation in vivo. The mutant receptor was active as a tyrosine kinase in vitro and in vivo, underwent CSF-1-dependent association with a phosphatidylinositol 3-kinase, and induced expression of the protooncogenes c-fos and junB, underscoring its ability to trigger some of the known cellular responses to CSF-1. The mutant receptor is likely to be impaired in its ability to interact with critical cellular effectors whose activity is required for mitogenesis.

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

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2domains reveal that the (HhH)2domains 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)2domains. 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)2domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  15. 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

  16. Characterization of the human granulocyte-macrophage colony-stimulating factor gene promoter: an AP1 complex and an Sp1-related complex transactivate the promoter activity that is suppressed by a YY1 complex.

    PubMed Central

    Ye, J; Zhang, X; Dong, Z

    1996-01-01

    It is well documented that a repeated CATT element in the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter is required for promoter activity. However, the transcription factors that are able to transactivate this enhancer element remain unidentified. Recently, we have found that nuclear factor YY1 can interact with the enhancer element. Here, we report that in addition to YY1, two other nuclear factors have been identified in the DNA-protein complexes formed by the CATT oligonucleotide and the Jurkat T-cell nuclear protein. One of these factors is AP1, and the other one is an Sp1-related protein. Results from transient transfection of Jurkat T cells have revealed that formation of both AP1 and the Sp1-related complex is required for the full enhancer activity of the CATT element. This result is supported by cotransfection of a c-jun expression vector and mutational analysis of the AP1 site or the Sp1-related protein binding site. In contrast, formation of the YY1 complex suppresses enhancer activity, since deletion of the YY1 complex induces an augmentation of the enhancer activity and overexpression of YY1 results in an attenuation of the enhancer activity. Results from the mechanism study have revealed that YY1 is able to inhibit transactivation mediated by either AP1 or the Sp1-related protein, and YY1 suppressive activity is DNA binding dependent. Taken together, these data support the ideas that AP1 and the Sp1-related nuclear protein are required for transactivation of the human GM-CSF gene promoter and that YY1 can suppress transactivation of the promoter even under inducible conditions. PMID:8524292

  17. Sox9 is a β-catenin-regulated transcription factor that enhances the colony-forming activity of squamous cell carcinoma cells.

    PubMed

    Li, Xue Mei; Piao, Yong Jun; Sohn, Kyung-Cheol; Ha, Jeong-Min; Im, Myung; Seo, Young-Joon; Whang, Kyu Uang; Lee, Jeung-Hoon; Lee, Young; Kim, Chang Deok

    2016-07-01

    Squamous cell carcinoma (SCC) is a common skin cancer, of which the incidence is relatively high, ranking second among the non‑melanoma skin cancers. It is known that numerous intracellular signal regulators are involved in the pathogenesis of SCC. The Wnt/β-catenin signaling pathway serves an important role in cancer development. However, the downstream effectors of β‑catenin remain to be clearly elucidated yet. The present study investigated the functional importance of Wnt/β‑catenin signaling in cutaneous SCC. β‑catenin expression was reduced using recombinant adenovirus expressing specific microRNA (miR). Knockdown of β‑catenin resulted in a marked reduction of the colony-forming activity of the SCC cells, SCC12. In an attempt to identify the β‑catenin downstream genes, it was found that Sox9 was regulated by β‑catenin in SCC12 cells. Overexpression of a constitutively active form of β‑catenin led to the induction of Sox9, while knockdown of β‑catenin resulted in downregulation of Sox9. When the expression of Sox9 was reduced using specific miR, colony-forming activity of the SCC12 cells was significantly reduced. When Sox9 was overexpressed in cells where β‑catenin was knocked down, it partially restored the colony‑forming potential. Taken together, the present results suggested that Sox9 is a β-catenin downstream transcription factor and is positively involved in SCC development. PMID:27151141

  18. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-01-01

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach. PMID:22374998

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

    SciTech Connect

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

    1991-10-01

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

  20. An active-site peptide from pepsin C

    PubMed Central

    Kay, J.; Ryle, A. P.

    1971-01-01

    Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed. PMID:4942834

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

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined. PMID:27243042

  2. Spread of plague among black-tailed prairie dogs is associated with colony spatial characteristics

    USGS Publications Warehouse

    Johnson, T.L.; Cully, J.F., Jr.; Collinge, S.K.; Ray, C.; Frey, C.M.; Sandercock, B.K.

    2011-01-01

    Sylvatic plague (Yersinia pestis) is an exotic pathogen that is highly virulent in black-tailed prairie dogs (Cynomys ludovicianus) and causes widespread colony losses and individual mortality rates >95%. We investigated colony spatial characteristics that may influence inter-colony transmission of plague at 3 prairie dog colony complexes in the Great Plains. The 4 spatial characteristics we considered include: colony size, Euclidean distance to nearest neighboring colony, colony proximity index, and distance to nearest drainage (dispersal) corridor. We used multi-state mark-recapture models to determine the relationship between these colony characteristics and probability of plague transmission among prairie dog colonies. Annual mapping of colonies and mark-recapture analyses of disease dynamics in natural colonies led to 4 main results: 1) plague outbreaks exhibited high spatial and temporal variation, 2) the site of initiation of epizootic plague may have substantially influenced the subsequent inter-colony spread of plague, 3) the long-term effect of plague on individual colonies differed among sites because of how individuals and colonies were distributed, and 4) colony spatial characteristics were related to the probability of infection at all sites although the relative importance and direction of relationships varied among sites. Our findings suggest that conventional prairie dog conservation management strategies, including promoting large, highly connected colonies, may need to be altered in the presence of plague. ?? 2011 The Wildlife Society.

  3. Multiple-factor influences upon feeding flight rates at wading bird colonies (Alias: Are flight-line counts useful?)

    USGS Publications Warehouse

    Erwin, R.M.; Ogden, J.C.

    1979-01-01

    The temporal patterns of feeding, resting, and reproductive behavior in colonial wading birds have been studied by a number of investigators, both on a short-term (daily) and long-term (annual) basis. In coastal marine environments, activities at colonies are influenced by tides, time of day and phase of the nesting cycle. The purpose of this paper is twofold: (1) to examine the effects of tide, time of day (physical factors), nesting phase, colony site, and species identity (biological factors) on feeding flight rates at breeding colonies and, as a result of this, (2) to evaluate the usefulness of feeding flight counts as an index of the number of nests in the colony. Earlier work suggests that the relationship between the number of individuals flying to and from the nesting colony may be quite consistent with nest numbers. Thus, by monitoring flights from remote locations, observers might obtain relatively accurate census data while minimizing time and disturbance at colonies. Recent concern for the deleterious impact of humans at waterbird colonies underscores the need to investigate alternative census methods.

  4. Multiscale habitat selection by burrowing owls in black-tailed prairie dog colonies

    USGS Publications Warehouse

    Lantz, S.J.; Conway, C.J.; Anderson, S.H.

    2007-01-01

    Some populations of western burrowing owls (Athene cunicularia hypugaea) have declined in recent decades. To design and implement effective recovery efforts, we need a better understanding of how distribution and demographic traits are influenced by habitat quality. To this end, we measured spatial patterns of burrowing owl breeding habitat selection within black-tailed prairie dog (Cynomys ludovicianus) colonies in northeastern Wyoming, USA. We compared burrow-, site-, colony-, and landscape-scale habitat parameters between burrowing owl nest burrows (n = 105) and unoccupied burrows (n = 85). We sampled 4 types of prairie dog colonies: 1) owl-occupied, active with prairie dogs (n = 16); 2) owl-occupied, inactive (n = 13); 3) owl-unoccupied, active (n = 14); and 4) owl-unoccupied, inactive (n = 14). We used an information-theoretic approach to examine a set of candidate models of burrowing owl nest-site selection. The model with the most support included variables at all 4 spatial scales, and results were consistent among the 4 types of prairie dog colonies. Nest burrows had longer tunnels, more available burrows within 30 m, and less shrub cover within 30 m, more prairie dog activity within 100 m, and were closer to water than unoccupied burrows. The model correctly classified 76% of cases, all model coefficients were stable, and the model had high predictive ability. Based on our results, we recommend actions to ensure persistence of the remaining prairie dog colonies as an important management strategy for burrowing owl conservation in the Great Plains of North America.

  5. The Toll-like receptor 4-activated neuroprotective microglia subpopulation survives via granulocyte macrophage colony-stimulating factor and JAK2/STAT5 signaling.

    PubMed

    Kamigaki, Mayumi; Hide, Izumi; Yanase, Yuhki; Shiraki, Hiroko; Harada, Kana; Tanaka, Yoshiki; Seki, Takahiro; Shirafuji, Toshihiko; Tanaka, Shigeru; Hide, Michihiro; Sakai, Norio

    2016-02-01

    Toll-like receptor (TLR) 4 mediates inflammation and is also known to trigger apoptosis in microglia. Our time-lapse observations showed that lipopolysaccharide (LPS) stimulation induced rapid death in primary cultures of rat microglia, while a portion of the microglia escaped from death and survived for much longer than 2 days, in which time, all of the control cells had died. However, it remains unclear how the LPS-stimulated microglia subpopulation could continue to survive in the absence of any supplied growth factors. In the present study, to clarify the mechanism underlying the LPS-stimulated survival, we investigated whether microglia could produce their own survival factors in response to LPS, focusing on macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-34, which are mainly supplied by astrocytes or neurons. The LPS-stimulated microglia drastically induced the expression of the GM-CSF mRNA and protein, while M-CSF and IL-34 levels were unchanged. The surviving microglia also significantly upregulated the expression of GM-CSF receptor (GM-CSFR) mRNA without affecting M-CSFR. As for the GM-CSFR downstream signal, LPS resulted in the phosphorylation of STAT5 and its translocation to the nucleus in the surviving microglia. Moreover, a specific JAK2 inhibitor, NVP-BSK805, suppressed STAT5 phosphorylation and microglia survival in response to LPS, indicating a critical role of the JAK2/STAT5 pathway in this survival mechanism. Together, these results suggest that a subpopulation of TLR4-activated microglia may survive by producing GM-CSF and up-regulating GM-CSFR. This autocrine GM-CSF pathway may activate the JAK2/STAT5 signaling pathway, which controls the transcription of survival-related genes. Finally, these surviving microglia may have neuroprotective functions because the neurons remained viable in co-cultures with these microglia. PMID:26802935

  6. Rat intestinal trehalase. Studies of the active site.

    PubMed

    Chen, C C; Guo, W J; Isselbacher, K J

    1987-11-01

    Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed. PMID:3426558

  7. Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

    PubMed Central

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; Burgos, Emmanuel S.; Gulab, Shivali A.; Schramm, Vern L.

    2015-01-01

    5′-Methylthioadenosine/S-adenosyl-L-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5′-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences. PMID:25806409

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  9. Registration of Colony Switchgrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Colony (Reg. No. __________, PI 658520) is a lowland cytotype of switchgrass (Panicum virgatum L.) developed and released by the USDA-ARS and North Carolina Agricultural Research Service. Colony was developed from three cycles of selection with random cross pollination occurring in cycle 0, index se...

  10. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  11. Energy transfer at the active sites of heme proteins

    SciTech Connect

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

    1995-12-31

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

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

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

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

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

    PubMed

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

    2016-09-01

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

  14. Active sites environmental monitoring program. Annual report FY 1992

    SciTech Connect

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

    1994-04-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) at ORNL from October 1991 through September 1992. Solid Waste Operations and the Environmental Sciences Division established ASEMP in 1989 to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by Chapter 2 and 3 of US Department of Energy Order 5820.2A. The Interim Waste Management Facility (IWMF) began operation in December 1991. Monitoring results from the tumulus and IWMF disposal pads continue to indicate that no LLW is leaching from the storage vaults. Storm water falling on the IWMF active pad was collected and transported to the Process Waste Treatment Plant while operators awaited approval of the National Pollutant Discharge Elimination System (NPDES) permit. Several of the recent samples collected from the active IWMF pad had pH levels above the NPDES limit of 9.0 because of alkali leached from the concrete. The increase in gross beta activity has been slight; only 1 of the 21 samples collected contained activity above the 5.0 Bq/L action level. Automated sample-collection and flow-measurement equipment has been installed at IWMF and is being tested. The flume designed to electronically measure flow from the IWMF pads and underpads is too large to be of practical value for measuring most flows at this site. Modification of this system will be necessary. A CO{sub 2} bubbler system designed to reduce the pH of water from the pads is being tested at IWMF.

  15. Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization, and Pathogenicity.

    PubMed

    Wei, Wei; Xiong, Ying; Zhu, Wenjun; Wang, Nancong; Yang, Guogen; Peng, Fang

    2016-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK) signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homolog in Colletotrichum lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum. PMID:27536296

  16. Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization, and Pathogenicity

    PubMed Central

    Wei, Wei; Xiong, Ying; Zhu, Wenjun; Wang, Nancong; Yang, Guogen; Peng, Fang

    2016-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK) signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homolog in Colletotrichum lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum. PMID:27536296

  17. Dynamics of Indian Ocean Slavery Revealed through Isotopic Data from the Colonial Era Cobern Street Burial Site, Cape Town, South Africa (1750-1827).

    PubMed

    Kootker, Lisette M; Mbeki, Linda; Morris, Alan G; Kars, Henk; Davies, Gareth R

    2016-01-01

    The Dutch East India Company (VOC) intended the Cape of Good Hope to be a refreshment stop for ships travelling between the Netherlands and its eastern colonies. The indigenous Khoisan, however, did not constitute an adequate workforce, therefore the VOC imported slaves from East Africa, Madagascar and Asia to expand the workforce. Cape Town became a cosmopolitan settlement with different categories of people, amongst them a non-European underclass that consisted of slaves, exiles, convicts and free-blacks. This study integrated new strontium isotope data with carbon and nitrogen isotope results from an 18th-19th century burial ground at Cobern Street, Cape Town, to identify non-European forced migrants to the Cape. The aim of the study was to elucidate individual mobility patterns, the age at which the forced migration took place and, if possible, geographical provenance. Using three proxies, 87Sr/86Sr, δ13Cdentine and the presence of dental modifications, a majority (54.5%) of the individuals were found to be born non-locally. In addition, the 87Sr/86Sr data suggested that the non-locally born men came from more diverse geographic origins than the migrant women. Possible provenances were suggested for two individuals. These results contribute to an improved understanding of the dynamics of slave trading in the Indian Ocean world. PMID:27309532

  18. Dynamics of Indian Ocean Slavery Revealed through Isotopic Data from the Colonial Era Cobern Street Burial Site, Cape Town, South Africa (1750-1827)

    PubMed Central

    Morris, Alan G.; Kars, Henk; Davies, Gareth R.

    2016-01-01

    The Dutch East India Company (VOC) intended the Cape of Good Hope to be a refreshment stop for ships travelling between the Netherlands and its eastern colonies. The indigenous Khoisan, however, did not constitute an adequate workforce, therefore the VOC imported slaves from East Africa, Madagascar and Asia to expand the workforce. Cape Town became a cosmopolitan settlement with different categories of people, amongst them a non-European underclass that consisted of slaves, exiles, convicts and free-blacks. This study integrated new strontium isotope data with carbon and nitrogen isotope results from an 18th-19th century burial ground at Cobern Street, Cape Town, to identify non-European forced migrants to the Cape. The aim of the study was to elucidate individual mobility patterns, the age at which the forced migration took place and, if possible, geographical provenance. Using three proxies, 87Sr/86Sr, δ13Cdentine and the presence of dental modifications, a majority (54.5%) of the individuals were found to be born non-locally. In addition, the 87Sr/86Sr data suggested that the non-locally born men came from more diverse geographic origins than the migrant women. Possible provenances were suggested for two individuals. These results contribute to an improved understanding of the dynamics of slave trading in the Indian Ocean world. PMID:27309532

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

    PubMed

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

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. PMID:17539607

  20. Intraspecific Variation among Social Insect Colonies: Persistent Regional and Colony-Level Differences in Fire Ant Foraging Behavior.

    PubMed

    Bockoven, Alison A; Wilder, Shawn M; Eubanks, Micky D

    2015-01-01

    Individuals vary within a species in many ecologically important ways, but the causes and consequences of such variation are often poorly understood. Foraging behavior is among the most profitable and risky activities in which organisms engage and is expected to be under strong selection. Among social insects there is evidence that within-colony variation in traits such as foraging behavior can increase colony fitness, but variation between colonies and the potential consequences of such variation are poorly documented. In this study, we tested natural populations of the red imported fire ant, Solenopsis invicta, for the existence of colony and regional variation in foraging behavior and tested the persistence of this variation over time and across foraging habitats. We also reared single-lineage colonies in standardized environments to explore the contribution of colony lineage. Fire ants from natural populations exhibited significant and persistent colony and regional-level variation in foraging behaviors such as extra-nest activity, exploration, and discovery of and recruitment to resources. Moreover, colony-level variation in extra-nest activity was significantly correlated with colony growth, suggesting that this variation has fitness consequences. Lineage of the colony had a significant effect on extra-nest activity and exploratory activity and explained approximately half of the variation observed in foraging behaviors, suggesting a heritable component to colony-level variation in behavior. PMID:26197456

  1. Intraspecific Variation among Social Insect Colonies: Persistent Regional and Colony-Level Differences in Fire Ant Foraging Behavior

    PubMed Central

    Bockoven, Alison A.; Wilder, Shawn M.; Eubanks, Micky D.

    2015-01-01

    Individuals vary within a species in many ecologically important ways, but the causes and consequences of such variation are often poorly understood. Foraging behavior is among the most profitable and risky activities in which organisms engage and is expected to be under strong selection. Among social insects there is evidence that within-colony variation in traits such as foraging behavior can increase colony fitness, but variation between colonies and the potential consequences of such variation are poorly documented. In this study, we tested natural populations of the red imported fire ant, Solenopsis invicta, for the existence of colony and regional variation in foraging behavior and tested the persistence of this variation over time and across foraging habitats. We also reared single-lineage colonies in standardized environments to explore the contribution of colony lineage. Fire ants from natural populations exhibited significant and persistent colony and regional-level variation in foraging behaviors such as extra-nest activity, exploration, and discovery of and recruitment to resources. Moreover, colony-level variation in extra-nest activity was significantly correlated with colony growth, suggesting that this variation has fitness consequences. Lineage of the colony had a significant effect on extra-nest activity and exploratory activity and explained approximately half of the variation observed in foraging behaviors, suggesting a heritable component to colony-level variation in behavior. PMID:26197456

  2. Role of Granulocyte-Macrophage Colony-Stimulating Factor Signaling in Regulating Neutrophil Antifungal Activity and the Oxidative Burst During Respiratory Fungal Challenge.

    PubMed

    Kasahara, Shinji; Jhingran, Anupam; Dhingra, Sourabh; Salem, Anand; Cramer, Robert A; Hohl, Tobias M

    2016-04-15

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that plays a critical role in regulating myeloid cell host defense. In this study, we demonstrated that GM-CSF signaling plays an essential role in antifungal defense against Aspergillus fumigatus. Mice that lack the GM-CSF receptor β chain (GM-CSFRβ) developed invasive hyphal growth and exhibited impaired survival after pulmonary challenge with A. fumigatus conidia. GM-CSFRβ signaling regulated the recruitment of inflammatory monocytes to infected lungs, but not the recruitment of effector neutrophils. Cell-intrinsic GM-CSFRβ signaling mediated neutrophil and inflammatory monocyte antifungal activity, because lung GM-CSFRβ(-/-) leukocytes exhibited impaired conidial killing compared with GM-CSFRβ(+/+) counterparts in mixed bone marrow chimeric mice. GM-CSFRβ(-/-) neutrophils exhibited reduced (hydrogenated) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in vivo. Conversely, administration of recombinant GM-CSF enhanced neutrophil NADPH oxidase function, conidiacidal activity, and lung fungal clearance in A. fumigatus-challenged mice. Thus, our study illustrates the functional role of GM-CSFRβ signaling on lung myeloid cell responses against inhaled A. fumigatus conidia and demonstrates a benefit for systemic GM-CSF administration. PMID:26908736

  3. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

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

    USGS Publications Warehouse

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

    2000-01-01

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

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

    PubMed Central

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

    2000-01-01

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

  6. Exploration adjustment by ant colonies

    PubMed Central

    2016-01-01

    How do animals in groups organize their work? Division of labour, i.e. the process by which individuals within a group choose which tasks to perform, has been extensively studied in social insects. Variability among individuals within a colony seems to underpin both the decision over which tasks to perform and the amount of effort to invest in a task. Studies have focused mainly on discrete tasks, i.e. tasks with a recognizable end. Here, we study the distribution of effort in nest seeking, in the absence of new nest sites. Hence, this task is open-ended and individuals have to decide when to stop searching, even though the task has not been completed. We show that collective search effort declines when colonies inhabit better homes, as a consequence of a reduction in the number of bouts (exploratory events). Furthermore, we show an increase in bout exploration time and a decrease in bout instantaneous speed for colonies inhabiting better homes. The effect of treatment on bout effort is very small; however, we suggest that the organization of work performed within nest searching is achieved both by a process of self-selection of the most hard-working ants and individual effort adjustment. PMID:26909180

  7. Exploration adjustment by ant colonies.

    PubMed

    Doran, Carolina; Stumpe, Martin C; Sendova-Franks, Ana; Franks, Nigel R

    2016-01-01

    How do animals in groups organize their work? Division of labour, i.e. the process by which individuals within a group choose which tasks to perform, has been extensively studied in social insects. Variability among individuals within a colony seems to underpin both the decision over which tasks to perform and the amount of effort to invest in a task. Studies have focused mainly on discrete tasks, i.e. tasks with a recognizable end. Here, we study the distribution of effort in nest seeking, in the absence of new nest sites. Hence, this task is open-ended and individuals have to decide when to stop searching, even though the task has not been completed. We show that collective search effort declines when colonies inhabit better homes, as a consequence of a reduction in the number of bouts (exploratory events). Furthermore, we show an increase in bout exploration time and a decrease in bout instantaneous speed for colonies inhabiting better homes. The effect of treatment on bout effort is very small; however, we suggest that the organization of work performed within nest searching is achieved both by a process of self-selection of the most hard-working ants and individual effort adjustment. PMID:26909180

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  9. Active Sites Environmental Monitoring Program. FY 1993: Annual report

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

    1994-08-01

    This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993.

  10. Parasitic fauna of a yellow-legged gull colony in the island of Escombreras (South-eastern Mediterranean) in close proximity to a landfill site: potential effects on cohabiting species.

    PubMed

    Parejo, Sandra Hervías; Martínez-Carrasco, Carlos; Diaz, Julia I; Chitimia, Lidia; Ortiz, Juana; Mayo, Elvira; Ybáñez, Rocío Ruiz de

    2015-06-01

    We identified the ectoparasites and helminth fauna of yellow-legged gulls (Larus michahellis michahellis), breeding near to a solid waste landfill, and compared infection levels with those of other yellow-legged gull colonies. Moreover, we analysed correlations between parasites and sex and body condition of yellow-legged gulls, co-infections and the helminth community structure in order to propose the role of this species as reservoir of certain parasites. We also discuss the potential transmission of parasites between the yellow-legged gull and the endangered Audouin's gull, because interactions between these two species, such as kleptoparasitism and predation, occur frequently around colonies. The following species were recorded: Ornithodorus capensis (Arthropoda); Cosmocephalus obvelatus, Paracuaria adunca, Eucoleus contortus, Tetrameres skrjabini and Contracaecum sp. (Nematoda); Tetrabothrius cylindraceus (Cestoda); Acanthotrema armata, Cardiocephaloides longicollis and Ornithobilharzia intermedia (Digenea). Tetrabothrius cylindraceus, A. armata and O. capensis are new parasite records for this host. The dependence of yellow-legged-gulls on fishery discards is supported by the dominance of parasites transmitted through marine intermediate hosts with interest to fisheries in the study area. However, the shift in diet from natural resources to food derived from human activities seems not to affect the parasitic fauna of yellow-legged gull. Besides of direct physical contact between individuals in nesting and resting habitats, the high availability of fishery discards could increase the risk of Audouin's gulls to be infected by common parasites of yellow-legged gull. PMID:26203998

  11. Active sites in char gasification: Final technical report

    SciTech Connect

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

    1987-09-01

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

  12. The Colonial Adult Educator

    ERIC Educational Resources Information Center

    Long, Huey B.

    1975-01-01

    The adult evening schools of colonial America served a mixed clientele and provided instruction in a variety of subjects. Although most historians have described evening schoolmasters as incompetent frauds, research indicates that such descriptions are generally unjust and unfounded. (NHM)

  13. Robotic space colonies

    NASA Technical Reports Server (NTRS)

    Schenker, P.; Easter, R.; Rodriguez, G.

    2001-01-01

    This paper reviews recent advances in these technologies, with a particular focus on experimental state-of-the-art robot work crew system demonstrations at JPL, that are being conducted now to begin to realize the futuristic robotic colony vision.

  14. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve

    PubMed Central

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  15. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve.

    PubMed

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  16. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

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

    2016-05-01

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

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

    SciTech Connect

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

    2014-08-14

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

  18. Spatial patterns in ant colonies.

    PubMed

    Theraulaz, Guy; Bonabeau, Eric; Nicolis, Stamatios C; Solé, Ricard V; Fourcassié, Vincent; Blanco, Stéphane; Fournier, Richard; Joly, Jean-Louis; Fernández, Pau; Grimal, Anne; Dalle, Patrice; Deneubourg, Jean-Louis

    2002-07-23

    The origins of large-scale spatial patterns in biology have been an important source of theoretical speculation since the pioneering work by Turing (1952) on the chemical basis of morphogenesis. Knowing how these patterns emerge and their functional role is important to our understanding of the evolution of biocomplexity and the role played by self organization. However, so far, conclusive evidence for local activation-long-range inhibition mechanisms in real biological systems has been elusive. Here a well-defined experimental and theoretical analysis of the pattern formation dynamics exhibited by clustering behavior in ant colonies is presented. These experiments and a simple mathematical model show that these colonies do indeed use this type of mechanism. All microscopic variables have been measured and provide the first evidence, to our knowledge, for this type of self-organized behavior in complex biological systems, supporting early conjectures about its role in the organization of insect societies. PMID:12114538

  19. P21-activated protein kinase (PAK2)-mediated c-Jun phosphorylation at 5 threonine sites promotes cell transformation

    PubMed Central

    Li, Tingting; Zhang, Jishuai; Zhu, Feng; Wen, Weihong; Zykova, Tatyana; Li, Xiang; Liu, Kangdong; Peng, Cong; Ma, Weiya; Shi, Guozheng; Dong, Ziming; Bode, Ann M.; Dong, Zigang

    2011-01-01

    The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 regulates the expression of many genes and is involved in a variety of biological functions such as cell transformation, proliferation, differentiation and apoptosis. AP-1 activates a variety of tumor-related genes and therefore promotes tumorigenesis and malignant transformation. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK) 2. Our data showed that PAK2 binds and phosphorylates c-Jun at five threonine sites (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and ex vivo. Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. Mutation at all five c-Jun threonine sites phosphorylated by PAK2 decreased the transforming ability of JB6 cells. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. These results indicated that PAK2/c-Jun signaling plays an important role in EGF-induced cell proliferation and transformation. PMID:21177766

  20. HOST SPECIFICITY OF THE MICROSPORIDIAN PATHOGEN VAIRIMORPHA INVICTAE AT FIVE FIELD SITES WITH INFECTED SOLENOPSIS INVICTA FIRE ANT COLONIES IN NORTHERN ARGENTINA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The microsporidian pathogen Vairimorpha invictae is being evaluated for release in the United States as a potential classical or self-sustaining biological control agent for imported fire ants. We examined the host range of this pathogen at five sites in northern Argentina where Solenopsis invicta ...

  1. The influence of fire-coral colony size and agonistic behaviour of territorial damselfish on associated coral reef fish communities.

    PubMed

    Leal, Isabela Carolina Silva; de Araújo, Maria Elisabeth; da Cunha, Simone Rabelo; Pereira, Pedro Henrique Cipresso

    2015-07-01

    Branching hydrocorals from the genus Millepora play an important ecological role in South Atlantic reefs, where branching scleractinian corals are absent. Previous studies have shown a high proportion of reef fish species using branching fire-coral colonies as shelter, breeding, and feeding sites. However, the effects of Millepora spp. colony size and how the agonistic behaviour of a competitive damselfish affect the associated reef fish community are still unknown. The present study examined how fire-coral colony volume and the presence of a highly territorial and aggressive damselfish (Brazilian endemic Stegastes fuscus) affects the reef fish community associated with the fire-coral Millepora alcicornis. M. alcicornis colonies were surveyed from September 2012 to April 2013 at Tamandaré Reefs off Northeast Brazil. Our results show that the abundance and richness of coral associated fish was positively correlated with M. alcicornis coral colony volume. Additionally, behaviour of S. fuscus, the most abundant reef fish species found associated with fire-coral colonies (almost 57% of the fish community), was also influenced by fire-coral colony volume. There was a clear trend of increased agonistic behaviour and feeding on coral polyps as colony volume increased. This trend was reversed for the non-occupational swimming category, which decreased as M. alcicornis colony volume increased. Behavioural ontogenetic changes were also detected for S. fuscus individuals. Juveniles mainly showed two distinct behaviours: sheltered on coral branches and feeding on coral polyps. In contrast, adults presented greater equitability among the behavioural categories, mostly non-occupational swimming around coral colonies and agonistic behaviour. Lastly, S. fuscus individuals actively defended fire-coral colonies from intruders. A large number of agonistic interactions occurred against potential food competitors, which were mainly roving herbivores, omnivores, and sessile

  2. Florida atlas of breeding sites for herons and their allies: 1976-1978

    SciTech Connect

    Nesbitt, S.A.; Ogden, J.C.; Kale, H.W. II; Patty, B.W.; Rowse, L.A.

    1982-08-01

    The first atlas of heron colonies was published in 1978 (Osborn and Custer 1978) and covered the Atlantic coast from Maine to Florida for 1975 and 1976. This atlas is an update of heron colonies for the Atlantic coast of Florida and an initial heron colony coverage for the Florida gulf Coast and inland colonies, 1976 through 1978. Aerial surveys were conducted in April and mid-summer over the Florida peninsula, east of the Ochlockonee River. A total of 295 active nesting colonies, with 22 different species, were located in the three-year period. Colony locations are shown on 1:250,000 USGS maps and Florida county maps. Colony data forms follow each county map and include information on location, date of census, estimated number of nesting pairs, site description, nesting substrate and proximity to human development.

  3. Reproduction, mortality, and heavy metal concentrations in great blue herons from three colonies in Washington and Idaho

    USGS Publications Warehouse

    Blus, L.J.; Henny, C.J.; Anderson, A.; Fitzner, R.E.

    1985-01-01

    We collected eggs in nests, hatchlings and eggs with advanced embryos on the ground, and prefledgling young of Great Blue Herons (Ardea herodias) at three nesting colonies in Washington and Idaho. Intact fish were also collected on the ground at the Idaho colony. The Ft. Lewis colony near Puget Sound in Washington and the Lake Chatcolet colony in northern Idaho were located near areas extensively polluted with heavy metals from minning or smelting activities. The Hanford Reservation colony near Richland, Washington was located some distance from point sources of heavy metal pollution. Heavy metals in heron samples were generally low and were all below concentrations known to induce mortality or adversely affect reproductive success. The elevated copper in one of three prefledglings from Ft. Lewis paralleled that found in an occasional nestling of several species of birds in other studies; the significance of this relationship is unclear. Breeding herons apparently fed near their colonies in areas removed from the sites of heaviest contamination, but birds in the Lake Chatcolet colony were preying on fish containing as much as 6 mu-g/g lead.

  4. Effect of inoculum size and water activity on the time to visible growth of Penicillium chrysogenum colony.

    PubMed

    Burgain, Anaïs; Bensoussan, Maurice; Dantigny, Philippe

    2013-05-15

    In order to assess the effect of the inoculum size on the time to visible growth for Penicillium chrysogenum, the correlation described by González et al. (González, H.H.L., Resnik, S.L., Vaamonde, G., 1987. Influence of inoculum size on growth rate and lag phase of fungi isolate from Argentine corn. International Journal of Food Microbiology 4, 111-117) was compared to the model introduced by Gougouli et al. (Gougouli, M., Kalantzi, K., Beletsiotis, E., Koutsoumanis, K.P., 2011. Development and application of predictive models for fungal growth as tools to improve quality control in yogurt production. Food Microbiology 28, 1453-1462). Based on the regression coefficient, the latter model performed better than the former one to fit the data obtained for P. chrysogenum grown on Potato Dextrose Agar at 25 °C. Inoculum sizes in the range 10(1)-10(5) spores were tested at 0.930, 0.950, 0.970, and 0.995 aw. By extrapolation of the straight line, the model of Gougouli et al. (2011) provided accurate estimations of the time to visible growth for a single spore inoculum, tvg (N=1). In order to avoid experiments at reduced water activities, the influence of water activity on the model parameters, and on the ratio tvg (N=1) over the germination time was assessed. PMID:23562694

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

    SciTech Connect

    1981-02-27

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

  6. Scaling of Traction Stresses with Size of Cohesive Cell Colonies

    NASA Astrophysics Data System (ADS)

    Mertz, Aaron F.; Banerjee, Shiladitya; Che, Yonglu; Marchetti, M. Christina; Horsley, Valerie; Dufresne, Eric R.

    2012-02-01

    We explore the mechanical properties of colonies of cohesive cells adherent on soft substrates. Specifically, we image the spatial distribution of traction stresses exerted by colonies of primary mouse keratinocytes on fibronectin-coated silicone gels. These cells have strong cell-cell adhesions mediated by E-cadherin. We observe that the work performed by a colony on its substrate is concentrated at the colony's periphery. The total work is strongly correlated to the geometrical size of the colony but not to number of cells. In other words, the mechanical output of a large single cell mimics that of a cohesive colony with the same overall size. We compare our findings to a recent theoretical model that treats the cohesive colony as an active gel.

  7. Wood Storks of the Birdsville Colony and swamps of the Savannah River Site: General overview of research findings, 1983--1990

    SciTech Connect

    Coulter, M.C.

    1993-02-01

    The population of Wood Storks (Mycteria americana) that breeds in the United States has decreased from an estimated 20,000 breeding pairs in 1930 to just under 5,000 pairs in 1980. Since 1980, the number has remained relatively stable, fluctuating between 3,500 and 5,500 breeding pairs. The decline prompted the US Fish and Wildlife Service (USFWS) to list the United States population of Wood Storks as endangered in 1984. When the US Department of Energy (USDOE) decided to restart L-Reactor on the Savannah River Site (SRS), there was concern that when the reactor was restarted, cooling water flowing into the Steel Creek Delta would raise the water level and the area would become too deep for foraging storks. The potential loss of this area to storks was important because storks had been observed foraging in the Steel Creek Delta. The USDOE began consultation with the USFWS in April, 1984, and the USDOE subsequently agreed to develop and maintain alternative foraging habitat to replace the potential loss. In order to design and manage the alternate foraging ponds as effectively as possible, it was necessary to understand aspects of the biology of the storks, the characteristics of their foraging sites and the patterns of their use of the SRSS. Results are described.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

    Hemsworth, Glyn R; Taylor, Edward J; Kim, Robbert Q; Gregory, Rebecca C; Lewis, Sally J; Turkenburg, Johan P; Parkin, Alison; Davies, Gideon J; Walton, Paul H

    2013-04-24

    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

  13. 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

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

    SciTech Connect

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

    2010-08-13

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

  15. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  16. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

    Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability. PMID:24086491

  17. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

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

    SciTech Connect

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

    2008-04-02

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

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

    SciTech Connect

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

    1986-05-01

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

  20. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  1. Eel calcitonin binding site distribution and antinociceptive activity in rats

    SciTech Connect

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

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

    SciTech Connect

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

    1992-02-01

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

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

    PubMed Central

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

    2015-01-01

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

  4. Active site and laminarin binding in glycoside hydrolase family 55.

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Women in Sports and Games in the Colonial Period.

    ERIC Educational Resources Information Center

    Howell, Reet A.

    The physical activities of women in the colonial period in the United States were limited. Social attitudes differed between the northern and southern colonies on appropriate activities for women. In the north it was not considered unseemly for women to participate in ice skating, while in the south women were encouraged to become good…

  7. Colonial American Astronomy

    NASA Astrophysics Data System (ADS)

    Yeomans, Donald K.

    2007-12-01

    While a foundation of German scientific methods enabled the rapid growth of North American Astronomy in the nineteenth century, during the seventeenth and most of the eighteenth centuries, the colonial men of science looked only to the English mother country for scientific patronage and guidance. An essay on fundamental astronomy appeared in one of the annual colonial almanacs as early as 1656, telescopic observations were made about 1660 and the first original colonial astronomical work was published by Thomas Danforth on the comet of 1664. By 1671 the Copernican ideas were so espoused at Harvard College that a physics class refused to read a Ptolemaic textbook when it was assigned to them by a senior instructor. At least in the Cambridge-Boston area, contemporary colonialist had access to the most recent scientific publications from the mother country. Observations of the great comet of 1680 by the Almanac maker, John Foster, reached Isaac Newton and were used and gratefully acknowledged in his Principia. During the seventeenth century the colonial interest in astronomy was more intense than it was for other sciences but colonists still occupied a position in the scientific backwater when compared with contemporary European scientists. Nevertheless, the science of astronomy was successfully transplanted from England to North America in the seventeenth century.

  8. The Colonial Inheritance.

    ERIC Educational Resources Information Center

    Allen, Jack

    As the American nation developed through periods of rapid change and great cultural diversity, the American public school system served as a primary souce of national unity. The roots of comprehensive public education in America can be traced to the educational system developed by 17th century Puritan colonials. Although one of the central…

  9. Abundance of diurnal raptors in relation to prairie dog colonies: Implications for bird-aircraft strike hazard

    USGS Publications Warehouse

    Merriman, J.W.; Boal, C.W.; Bashore, T.L.; Zwank, P.J.; Wester, D.B.

    2007-01-01

    Some diurnal raptors are frequently observed at prairie dog (Cynomys sp.) colonies. As a result, some military installations have conducted prairie dog control activities to reduce the bird-aircraft strike hazard (BASH) potential of low-flying aircraft. To evaluate the validity of this management strategy, we assessed raptor associations with prairie dog colonies at 2 short-grass prairie study areas: southern Lubbock County, Texas, USA, and Melrose Bombing and Gunnery Range in east-central New Mexico, USA. We quantified diurnal raptors (i.e., Falconiformes) at plots occupied (colony plots) and unoccupied (noncolony plots) by black-tailed prairie dogs (Cynomys ludovicianus) at both sites throughout 2002. We compared the number of individual birds of a given species at colony and noncolony plots within each study area by season. Ferruginous hawks (Buteo regalis) and northern harriers (Circus cyaneus) were more abundant at colony plots, whereas Swainson's hawks (B. swainsoni) and American kestrels (Falco sparverius) were more abundant at noncolony plots. Red-tailed hawk (B. jamaicensis) abundance did not differ between the 2 plot types. Our results suggest prairie dog control as a method of reducing BASH potential may be effective at some sites but may be ineffective or even increase the BASH potential at others. Thus, bird-avoidance models assessing the BASH potential should be conducted on a site-specific basis using information on relative and seasonal abundances of individual raptor species and the relative strike risks they pose to aircraft.

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

    PubMed

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

    2015-01-01

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

  11. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  12. Response of mountain plovers to plague-driven dynamics of black-tailed prairie dog colonies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sylvatic plague is a major factor influencing prairie dog colony dynamics in the western Great Plains. We studied the nesting response of the mountain plover (Charadrius montanus), a grassland bird that nests on prairie dog colonies, to plague-driven dynamics of prairie dog colonies at three sites i...

  13. Synergistic action of the benzene metabolite hydroquinone on myelopoietic stimulating activity of granulocyte/macrophage colony-stimulating factor in vitro

    NASA Technical Reports Server (NTRS)

    Irons, R. D.; Stillman, W. S.; Colagiovanni, D. B.; Henry, V. A.; Clarkson, T. W. (Principal Investigator)

    1992-01-01

    The effects of in vitro pretreatment with benzene metabolites on colony-forming response of murine bone marrow cells stimulated with recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) were examined. Pretreatment with hydroquinone (HQ) at concentrations ranging from picomolar to micromolar for 30 min resulted in a 1.5- to 4.6-fold enhancement in colonies formed in response to rGM-CSF that was due to an increase in granulocyte/macrophage colonies. The synergism equaled or exceeded that reported for the effects of interleukin 1, interleukin 3, or interleukin 6 with GM-CSF. Optimal enhancement was obtained with 1 microM HQ and was largely independent of the concentration of rGM-CSF. Pretreatment with other authentic benzene metabolites, phenol and catechol, and the putative metabolite trans, trans-muconaldehyde did not enhance growth factor response. Coadministration of phenol and HQ did not enhance the maximal rGM-CSF response obtained with HQ alone but shifted the optimal concentration to 100 pM. Synergism between HQ and rGM-CSF was observed with nonadherent bone marrow cells and lineage-depleted bone marrow cells, suggesting an intrinsic effect on recruitment of myeloid progenitor cells not normally responsive to rGM-CSF. Alterations in differentiation in a myeloid progenitor cell population may be of relevance in the pathogenesis of acute myelogenous leukemia secondary to drug or chemical exposure.

  14. Synergistic action of the benzene metabolite hydroquinone on myelopoietic stimulating activity of granulocyte/macrophage colony-stimulating factor in vitro.

    PubMed Central

    Irons, R D; Stillman, W S; Colagiovanni, D B; Henry, V A

    1992-01-01

    The effects of in vitro pretreatment with benzene metabolites on colony-forming response of murine bone marrow cells stimulated with recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) were examined. Pretreatment with hydroquinone (HQ) at concentrations ranging from picomolar to micromolar for 30 min resulted in a 1.5- to 4.6-fold enhancement in colonies formed in response to rGM-CSF that was due to an increase in granulocyte/macrophage colonies. The synergism equaled or exceeded that reported for the effects of interleukin 1, interleukin 3, or interleukin 6 with GM-CSF. Optimal enhancement was obtained with 1 microM HQ and was largely independent of the concentration of rGM-CSF. Pretreatment with other authentic benzene metabolites, phenol and catechol, and the putative metabolite trans, trans-muconaldehyde did not enhance growth factor response. Coadministration of phenol and HQ did not enhance the maximal rGM-CSF response obtained with HQ alone but shifted the optimal concentration to 100 pM. Synergism between HQ and rGM-CSF was observed with nonadherent bone marrow cells and lineage-depleted bone marrow cells, suggesting an intrinsic effect on recruitment of myeloid progenitor cells not normally responsive to rGM-CSF. Alterations in differentiation in a myeloid progenitor cell population may be of relevance in the pathogenesis of acute myelogenous leukemia secondary to drug or chemical exposure. PMID:1570288

  15. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  16. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

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

    PubMed

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

    2015-05-01

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

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

    PubMed

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

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth. PMID:26360629

  19. Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase.

    PubMed

    Li, Chi-Hui; Tu, Shiao-Chun

    2005-10-01

    Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed. PMID:16185065

  20. Growing Yeast into Cylindrical Colonies

    PubMed Central

    Vulin, Clément; Di Meglio, Jean-Marc; Lindner, Ariel B.; Daerr, Adrian; Murray, Andrew; Hersen, Pascal

    2014-01-01

    Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes. PMID:24853750

  1. Information use in colonial living.

    PubMed

    Evans, Julian C; Votier, Stephen C; Dall, Sasha R X

    2016-08-01

    Despite the fact that many animals live in groups, there is still no clear consensus about the ecological or evolutionary mechanisms underlying colonial living. Recently, research has suggested that colonies may be important as sources of social information. The ready availability of information from conspecifics allows animals to make better decisions about avoiding predators, reducing brood parasitism, migratory phenology, mate choice, habitat choice and foraging. These choices can play a large part in the development and maintenance of colonies. Here we review the types of information provided by colonial animals and examine the different ways in which decision-making in colonies can be enhanced by social information. We discuss what roles information might take in the evolution, formation and maintenance of colonies. In the process, we illustrate that information use permeates all aspects of colonial living. PMID:25882618

  2. The toothless osteopetrotic rat has a normal vitamin D-binding protein-macrophage activating factor (DBP-MAF) cascade and chondrodysplasia resistant to treatments with colony stimulating factor-1 (CSF-1) and/or DBP-MAF.

    PubMed

    Odgren, P R; Popoff, S N; Safadi, F F; MacKay, C A; Mason-Savas, A; Seifert, M F; Marks, S C

    1999-08-01

    The osteopetrotic rat mutation toothless (tl) is characterized by little or no bone resorption, few osteoclasts and macrophages, and chondrodysplasia at the growth plates. Short-term treatment of tl rats with colony-stimulating factor-1 (CSF-1) has been shown to increase the number of osteoclasts and macrophages, producing dramatic resolution of skeletal sclerosis at some, but not all, sites. Defects in production of vitamin D-binding protein-macrophage activating factor (DBP-MAF) have been identified in two other independent osteopetrotic mutations of the rat (op and ia), and two in the mouse (op and mi), in which macrophages and osteoclasts can be activated by the administration of exogenous DBP-MAF. The present studies were undertaken to examine the histology and residual growth defects in tl rats following longer CSF-1 treatments, to investigate the possibility that exogenous DBP-MAF might act synergistically with CSF-1 to improve the tl phenotype, and to assess the integrity of the endogenous DBP-MAF pathway in this mutation. CSF-1 treatment-with or without DBP-MAF-induced resorption of metaphyseal bone to the growth plate on the marrow side, improved slightly but did not normalize long bone growth, and caused no improvement in the abnormal histology of the growth plate. Injections of lysophosphatidylcholine (lyso-Pc) to prime macrophage activation via the DBP-MAF pathway raised superoxide production to similar levels in peritoneal macrophages from both normal and mutant animals, indicating no defect in the DBP-MAF pathway in tl rats. Interestingly, pretreatments with CSF-1 alone also increased superoxide production, although the mechanism for this remains unknown. In summary, we find that, unlike other osteopetrotic mutations investigated to date, the DBP-MAF pathway does not appear to be defective in the tl rat; that additional DBP-MAF does not augment the beneficial skeletal effects seen with CSF-1 alone; and that the growth plate chondrodystrophy seen in

  3. Parasitism and phenotypic change in colonial hosts.

    PubMed

    Hartikainen, Hanna; Fontes, Inês; Okamura, Beth

    2013-09-01

    Changes in host phenotype are often attributed to manipulation that enables parasites to complete trophic transmission cycles. We characterized changes in host phenotype in a colonial host–endoparasite system that lacks trophic transmission (the freshwater bryozoan Fredericella sultana and myxozoan parasite Tetracapsuloides bryosalmonae). We show that parasitism exerts opposing phenotypic effects at the colony and module levels. Thus, overt infection (the development of infectious spores in the host body cavity) was linked to a reduction in colony size and growth rate, while colony modules exhibited a form of gigantism. Larger modules may support larger parasite sacs and increase metabolite availability to the parasite. Host metabolic rates were lower in overtly infected relative to uninfected hosts that were not investing in propagule production. This suggests a role for direct resource competition and active parasite manipulation (castration) in driving the expression of the infected phenotype. The malformed offspring (statoblasts) of infected colonies had greatly reduced hatching success. Coupled with the severe reduction in statoblast production this suggests that vertical transmission is rare in overtly infected modules. We show that although the parasite can occasionally infect statoblasts during overt infections, no infections were detected in the surviving mature offspring, suggesting that during overt infections, horizontal transmission incurs a trade-off with vertical transmission. PMID:23965820

  4. Agricultural Societies in Colonial Western Australia in 1831-70.

    ERIC Educational Resources Information Center

    White, Michael

    2000-01-01

    Reviews the knowledge-diffusion activities of colonial agricultural societies in western Australia from their foundation in 1831-1870. States that from 1829 to 1850 British settlers belonged to a society of free citizens, while from 1851-1867 the settlement changed to a convict colony. (CMK)

  5. Colony image acquisition and genetic segmentation algorithm and colony analyses

    NASA Astrophysics Data System (ADS)

    Wang, W. X.

    2012-01-01

    Colony anaysis is used in a large number of engineerings such as food, dairy, beverages, hygiene, environmental monitoring, water, toxicology, sterility testing. In order to reduce laboring and increase analysis acuracy, many researchers and developers have made efforts for image analysis systems. The main problems in the systems are image acquisition, image segmentation and image analysis. In this paper, to acquire colony images with good quality, an illumination box was constructed. In the box, the distances between lights and dishe, camra lens and lights, and camera lens and dishe are adjusted optimally. In image segmentation, It is based on a genetic approach that allow one to consider the segmentation problem as a global optimization,. After image pre-processing and image segmentation, the colony analyses are perfomed. The colony image analysis consists of (1) basic colony parameter measurements; (2) colony size analysis; (3) colony shape analysis; and (4) colony surface measurements. All the above visual colony parameters can be selected and combined together, used to make a new engineeing parameters. The colony analysis can be applied into different applications.

  6. A proposed definition of the 'activity' of surface sites on lactose carriers for dry powder inhalation.

    PubMed

    Grasmeijer, Floris; Frijlink, Henderik W; de Boer, Anne H

    2014-06-01

    A new definition of the activity of surface sites on lactose carriers for dry powder inhalation is proposed which relates to drug detachment during dispersion. The new definition is expected to improve the understanding of 'carrier surface site activity', which stimulates the unambiguous communication about this subject and may aid in the rational design and interpretation of future formulation studies. In contrast to the currently prevailing view on carrier surface site activity, it follows from the newly proposed definition that carrier surface site activity depends on more variables than just the physicochemical properties of the carrier surface. Because the term 'active sites' is ambiguous, it is recommended to use the term 'highly active sites' instead to denote carrier surface sites with a relatively high activity. PMID:24613490

  7. Leisure, economy and colonial urbanism: Darjeeling, 1835–1930

    PubMed Central

    BHATTACHARYA, NANDINI

    2013-01-01

    This article posits that the hill station of Darjeeling was a unique form of colonial urbanism. It shifts historiographical interest from major urban centres in colonial India (such as Bombay or Calcutta) and instead attempts a greater understanding of smaller urban centres. In the process, it also interrogates the category of hill stations, which have been understood as exotic and scenic sites rather than as towns that were integral to the colonial economy. In arguing that hill stations, particularly Darjeeling, were not merely the scenic and healthy ‘other’ of the clamorous, dirty and diseased plains of India, it refutes suggestions that the ‘despoiling’ or overcrowding of Darjeeling was incremental to the purposes of its establishment. Instead, it suggests that Darjeeling was part of the colonial mainstream; its urbanization and inclusion into the greater colonial economy was effected from the time of its establishment. Therefore, a constant tension between its exotic and its functional elements persisted throughout. PMID:24273391

  8. Seasonal activity of the small hive beetle, Aethina tumida, as estimated by baited flight traps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seasonal variation in flight activity of the small hive beetle was monitored at two sites in north-central Florida, one near colonies of the European honeybee and the other far removed from bee colonies. Activity was monitored by flight traps baited with fermenting pollen dough that had been inocul...

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

    PubMed

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

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. PMID:25727891

  10. Construction of DNA recognition sites active in Haemophilus transformation.

    PubMed Central

    Danner, D B; Smith, H O; Narang, S A

    1982-01-01

    Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer. Images PMID:6285382

  11. Characterization of active site residues of nitroalkane oxidase.

    PubMed

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

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  12. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  13. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  14. Response of Reticulitermes hesperus (Isoptera: Rhinotermitidae) colonies to baiting with lufenuron in northern California.

    PubMed

    Haverty, Michael I; Tabuchi, Robin L; Vargo, Edward L; Cox, David L; Nelson, Lori J; Lewis, Vernard R

    2010-06-01

    The objective of this study was to evaluate lufenuron termite bait (1,500 ppm) for the elimination of colonies of Reticulitermes hesperus Banks (Isoptera: Rhinotermitidae). Dispersion of colonies in six baited and six unbaited sites near Placerville, CA, was determined by genetic (microsatellite) analyses. Twenty-one colonies of R. hesperus inhabited the six baited sites and eight colonies of R. hesperus occurred in the six unbaited sites. Five criteria provided a cause-and-effect link between the deployment of lufenuron termite bait and elimination of baited colonies: 1) association of foragers, as members of the same colony, in the independent monitoring stations and bait stations; 2) quantity of bait consumed; 3) abnormal physical appearance of foragers in bait stations; 4) disappearance of foragers from, and cessation of feeding in, independent monitoring stations visited by baited colonies; and 5) presence of foragers from, and continuation of feeding in, independent monitors visited by unbaited colonies. Baited colonies were devoid of foraging termites within a mean of 70.6 d (range, 37-93 d) of bait deployment. Colonies consumed a mean of 8.0 g of bait (range, 2.2-16.0 g). Wood consumption by baited and unbaited colonies was not significantly different during the 2 mo before baiting, 281.4 versus 590.5 mg/d per colony, respectively, nor during the 3 mo immediately after baiting, 112.5 versus 436.8 mg/d per colony, respectively. However, from 10 to 16 mo after baiting, wood consumption by baited colonies essentially ceased and was significantly less than the unbaited colonies, 7.9 versus 470.1 mg/d per colony, respectively. PMID:20568623

  15. Influence of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), macrophage-colony stimulating factor (M-CSF) and fetal calf serum on human osteoclast formation and activity.

    PubMed

    Kreja, Ludwika; Liedert, Astrid; Schmidt, Carla; Claes, Lutz; Ignatius, Anita

    2007-10-01

    Human osteoclast (OC) formation and activity was studied in cultures of peripheral blood mononuclear cells (PBMNC) from six healthy donors after stimulation with fetal calf serum (FCS), under the influence of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and the macrophage-colony stimulating factor (M-CSF). The results showed that selected FCS could stimulate OC formation without any medium supplementation with osteoclastogenic factors. The OC formation, investigated by quantification of multinucleated tartrate-resistant acid phosphatase-positive cells (TRAP+ cells), and the sensitivity of OC progenitors to RANKL and M-CSF, varied widely between individual donors. The OC resorption activity, measured in the "pit-assay" on dentine, was strictly dependent on the presence of RANKL and M-CSF in the medium and was also donor dependent. The considerable donor variability should be considered in culture studies investigating, e.g. the interactions of OC with biomaterials or the influence of cytokines, growth factors and drugs on osteoclastogenesis. PMID:18161075

  16. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  17. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

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

  18. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

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

  19. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

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

  20. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

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

  1. Fever in honeybee colonies

    NASA Astrophysics Data System (ADS)

    Starks, P. T.; Blackie, Caroline A.; Seeley, Thomas D.

    Honeybees, Apis spp., maintain elevated temperatures inside their nests to accelerate brood development and to facilitate defense against predators. We present an additional defensive function of elevating nest temperature: honeybees generate a brood-comb fever in response to colonial infection by the heat-sensitive pathogen Ascosphaera apis. This response occurs before larvae are killed, suggesting that either honeybee workers detect the infection before symptoms are visible, or that larvae communicate the ingestion of the pathogen. This response is a striking example of convergent evolution between this "superorganism" and other fever-producing animals.

  2. Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity.

    PubMed

    Rossetto, O; Caccin, P; Rigoni, M; Tonello, F; Bortoletto, N; Stevens, R C; Montecucco, C

    2001-08-01

    Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT. PMID:11306125

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

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

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

  4. Dynamically Achieved Active Site Precision in Enzyme Catalysis

    PubMed Central

    2015-01-01

    Conspectus The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes’ enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme–substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed. PMID:25539048

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

    SciTech Connect

    Teese, G.D.

    1995-09-28

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

  6. Colony image acquisition and segmentation

    NASA Astrophysics Data System (ADS)

    Wang, W. X.

    2007-12-01

    For counting of both colonies and plaques, there is a large number of applications including food, dairy, beverages, hygiene, environmental monitoring, water, toxicology, sterility testing, AMES testing, pharmaceuticals, paints, sterile fluids and fungal contamination. Recently, many researchers and developers have made efforts for this kind of systems. By investigation, some existing systems have some problems. The main problems are image acquisition and image segmentation. In order to acquire colony images with good quality, an illumination box was constructed as: the box includes front lightning and back lightning, which can be selected by users based on properties of colony dishes. With the illumination box, lightning can be uniform; colony dish can be put in the same place every time, which make image processing easy. The developed colony image segmentation algorithm consists of the sub-algorithms: (1) image classification; (2) image processing; and (3) colony delineation. The colony delineation algorithm main contain: the procedures based on grey level similarity, on boundary tracing, on shape information and colony excluding. In addition, a number of algorithms are developed for colony analysis. The system has been tested and satisfactory.

  7. Improving upon Nature: Active site remodeling produces highly efficient aldolase activity towards hydrophobic electrophilic substrates

    PubMed Central

    Cheriyan, Manoj; Toone, Eric J.; Fierke, Carol A.

    2012-01-01

    Substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S/S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2′-pyridyl)butyrate (S-KHPB). These mutations improve the value of kcat/KMS-KHPB by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of kcatS-KHPB for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximum value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate (KHO), a non-functionalized hydrophobic analog. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases. Furthermore, targeting mutations to the active site provides marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to naturally occurring enzymes. PMID

  8. Atomically-thin two-dimensional sheets for understanding active sites in catalysis.

    PubMed

    Sun, Yongfu; Gao, Shan; Lei, Fengcai; Xie, Yi

    2015-02-01

    Catalysis can speed up chemical reactions and it usually occurs on the low coordinated steps, edges, terraces, kinks and corner atoms that are often called "active sites". However, the atomic level interplay between active sites and catalytic activity is still an open question, owing to the large difference between idealized models and real catalysts. This stimulates us to pursue a suitable material model for studying the active sites-catalytic activity relationship, in which the atomically-thin two-dimensional sheets could serve as an ideal model, owing to their relatively simple type of active site and the ultrahigh fraction of active sites that are comparable to the overall atoms. In this tutorial review, we focus on the recent progress in disclosing the factors that affect the activity of reactive sites, including characterization of atomic coordination number, structural defects and disorder in ultrathin two-dimensional sheets by X-ray absorption fine structure spectroscopy, positron annihilation spectroscopy, electron spin resonance and high resolution transmission electron microscopy. Also, we overview their applications in CO catalytic oxidation, photocatalytic water splitting, electrocatalytic oxygen and hydrogen evolution reactions, and hence highlight the atomic level interplay among coordination number, structural defects/disorder, active sites and catalytic activity in the two-dimensional sheets with atomic thickness. Finally, we also present the major challenges and opportunities regarding the role of active sites in catalysis. We believe that this review provides critical insights for understanding the catalysis and hence helps to develop new catalysts with high catalytic activity. PMID:25382246

  9. The active sites of supported silver particle catalysts in formaldehyde oxidation.

    PubMed

    Chen, Yaxin; Huang, Zhiwei; Zhou, Meijuan; Hu, Pingping; Du, Chengtian; Kong, Lingdong; Chen, Jianmin; Tang, Xingfu

    2016-08-01

    Surface silver atoms with upshifted d-orbitals are identified as the catalytically active sites in formaldehyde oxidation by correlating their activity with the number of surface silver atoms, and the degree of the d-orbital upshift governs the catalytic performance of the active sites. PMID:27406403

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

    PubMed Central

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

    2014-01-01

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

  11. Small mammals associated with colonies of black-tailed prairie dogs (Cynomys ludovicianus) in the Southern High Plains

    USGS Publications Warehouse

    Pruett, A.L.; Boal, C.W.; Wallace, M.C.; Whitlaw, H.A.; Ray, J.D.

    2010-01-01

    We compared diversity and abundance of small mammals at colonies of black-tailed prairie dogs (Cynomys ludovicianus) and paired non-colony sites. Of colonies of black-tailed prairie dogs in our study area, >80 were on slopes of playa lakes; thus, we used sites of colonies and non-colonies that were on slopes of playa lakes. We trapped small mammals on 29 pairs of sites. Overall abundance did not differ between types of sites, but some taxa exhibited associations with colonies (Onychomys leucogaster) or non-colonies (Chaetodipus hispidus, Reithrodontomys, Sigmodon hispidus). Diversity and evenness of small mammals did not differ between colonies and non-colonies in 2002, but were higher on non-colonies in 2003. Although we may not have detected some rare or infrequently occurring species, our data reveal differences in diversity and evenness of more common species among the types of sites. Prairie dogs are touted as a keystone species with their colonies associated with a greater faunal diversity than adjacent lands. Our findings contradict several studies reporting greater diversity and abundance of small mammals at colonies of prairie dogs. We suggest that additional research across a wider landscape and incorporating landscape variables beyond the immediate trapping plot may further elucidate interspecific associations between black-tailed prairie dogs and species of small rodents.

  12. Post-Colonial Theory and Action Research

    ERIC Educational Resources Information Center

    Parsons, Jim B.; Harding, Kelly J.

    2011-01-01

    This essay explores connections between post-colonial theory and action research. Post-colonial theory is committed to addressing the plague of colonialism. Action research, at its core, promises to problematize uncontested "colonial" hegemonies of any form. Both post-colonial theory and action research engage dialogic, critically reflective and…

  13. Student Discipline in Colonial America.

    ERIC Educational Resources Information Center

    Petry, John R.

    The basis for the severe discipline imposed on school children in colonial America, especially in the Puritan colonies, was the belief in original sin. The child was regarded as being born in sin and thus depraved and prone to sin. The purpose of education was to enable children to read the Bible and thus change the behavior which otherwise would…

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

    PubMed Central

    Stec, Boguslaw

    2012-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. PMID:23112176

  15. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

    ... identified Wind Energy Area (WEA) on the OCS offshore Rhode Island (RI) and Massachusetts (MA). The revised... from leasing, site characterization, and site assessment in and around the Call Area (76 FR 51391). The... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  16. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ... specific project proposals on those leases) in an identified Wind Energy Area (WEA) on the OCS offshore..., site characterization, and site assessment in and around the Call Area (76 FR 51391). The Call Area is... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

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

    DOE Data Explorer

    John Peterson

    2015-04-17

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

  18. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

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

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

    SciTech Connect

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

    1994-09-01

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

  20. Ammonia emissions from seabird colonies

    NASA Astrophysics Data System (ADS)

    Blackall, Trevor D.; Wilson, Linda J.; Theobald, Mark R.; Milford, Celia; Nemitz, Eiko; Bull, Jennifer; Bacon, Philip J.; Hamer, Keith C.; Wanless, Sarah; Sutton, Mark A.

    2007-05-01

    Ammonia emissions were measured from two entire seabird colonies with contrasting species assemblages, to ascertain the ammonia volatilisation potentials among seabird species in relation to their nesting behaviour. Emissions were calculated from downwind plume measurements of ammonia concentration using both inverse dispersion and tracer ratio methods. Measured colony emissions ranged 1-90 kg NH3 hour-1, and equated to 16 and 36% volatilization of excreted nitrogen for colonies dominated by ground/burrow nesting and bare rock nesting birds, respectively. The results were applied in a bioenergetics model with a global seabird database. Seabird colonies are found to represent the largest point sources of ammonia globally (up to ~6 Gg NH3 colony-1 year-1). Moreover the largest emissions occur mainly in remote environments with otherwise low NH3 emissions. These ammonia ``hot spots'' explain significant perturbations of the nitrogen cycle in these regions and add ~20% to oceanic ammonia emissions south of latitude 45°S.

  1. Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

    This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

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

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

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

  3. Colony variation in the collective regulation of foraging by harvester ants

    PubMed Central

    Guetz, Adam; Greene, Michael J.; Holmes, Susan

    2011-01-01

    This study investigates variation in collective behavior in a natural population of colonies of the harvester ant, Pogonomyrmex barbatus. Harvester ant colonies regulate foraging activity to adjust to current food availability; the rate at which inactive foragers leave the nest on the next trip depends on the rate at which successful foragers return with food. This study investigates differences among colonies in foraging activity and how these differences are associated with variation among colonies in the regulation of foraging. Colonies differ in the baseline rate at which patrollers leave the nest, without stimulation from returning ants. This baseline rate predicts a colony's foraging activity, suggesting there is a colony-specific activity level that influences how quickly any ant leaves the nest. When a colony's foraging activity is high, the colony is more likely to regulate foraging. Moreover, colonies differ in the propensity to adjust the rate of outgoing foragers to the rate of forager return. Naturally occurring variation in the regulation of foraging may lead to variation in colony survival and reproductive success. PMID:22479133

  4. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts.

    PubMed

    Wang, Lu-Cun; Friend, C M; Fushimi, Rebecca; Madix, Robert J

    2016-07-01

    The activation of molecular O2 as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O2 activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O2 dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O2 dissociation is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O2 dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction. PMID:27376884

  5. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein. PMID:26620444

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

    PubMed Central

    2015-01-01

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

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

    SciTech Connect

    Not Available

    1993-01-27

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

  8. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.

    PubMed

    Kapetanaki, Sofia M; Field, Sarah J; Hughes, Ross J L; Watmough, Nicholas J; Liebl, Ursula; Vos, Marten H

    2008-01-01

    The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site. PMID:18420024

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

    PubMed Central

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

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

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

    PubMed

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

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  11. SEASON OF DELTAMETHRIN APPLICATION AFFECTS FLEA AND PLAGUE CONTROL IN WHITE-TAILED PRAIRIE DOG (CYNOMYS LEUCURUS) COLONIES, COLORADO, USA.

    PubMed

    Tripp, Daniel W; Streich, Sean P; Sack, Danielle A; Martin, Daniel J; Griffin, Karen A; Miller, Michael W

    2016-07-01

    In 2008 and 2009, we evaluated the duration of prophylactic deltamethrin treatments in white-tailed prairie dog ( Cynomys leucurus ) colonies and compared effects of autumn or spring dust application in suppressing flea numbers and plague. Plague occurred before and during our experiment. Overall, flea abundance tended to increase from May or June to September, but it was affected by deltamethrin treatment and plague dynamics. Success in trapping prairie dogs (animals caught/trap days) declined between June and September at all study sites. However, by September trap success on dusted sites (19%; 95% confidence interval [CI] 16-22%) was about 15-fold greater than on undusted control sites (1%; CI 0.3-4%; P≤0.0001). Applying deltamethrin dust as early as 12 mo prior seemed to afford some protection to prairie dogs. Our data showed that dusting even a portion of a prairie dog colony can prolong its persistence despite epizootic plague. Autumn dusting may offer advantages over spring in suppressing overwinter or early-spring flea activity, but timing should be adjusted to precede the annual decline in aboveground activity for hibernating prairie dog species. Large colony complexes or collections of occupied but fragmented habitat may benefit from dusting some sites in spring and others in autumn to maximize flea suppression in a portion of the complex or habitat year-round. PMID:27195680

  12. Predation of artificial ground nests on white-tailed prairie dog colonies

    USGS Publications Warehouse

    Baker, B.W.; Stanley, T.R.; Sedgwick, J.A.

    1999-01-01

    Prairie dog (Cynomys spp.) colonies are unique to prairie and shrub-steppe landscapes. However, widespread eradication, habitat loss, and sylvatic plague (Yersinia pestis) have reduced their numbers by 98% since historical times. Birds associated with prairie dogs also are declining. Potential nest predators, such as coyotes (Canis latrans), swift foxes (Vulpes velox), and badgers (Taxidea taxus), may be attracted to colonies where a high concentration of prairie dogs serve as available prey. Increased abundance of small mammals, including prairie dogs, also may increase the risk of predation for birds nesting on colonies. Finally, because grazing by prairie dogs may decrease vegetation height and canopy cover, bird nests may be easier for predators to locate. In this study, we placed 1,444 artificial ground nests on and off 74 white-tailed prairie dog (C. leucurus) colonies to test the hypothesis that nest predation rates are higher on colonies than at nearby off sites (i.e., uncolonized habitat). We sampled colonies from 27 May to 16 July 1997 at the following 3 complexes: Coyote Basin, Utah and Colorado; Moxa Arch, Wyoming; and Shirley Basin, Wyoming. Differences in daily predation rates between colonies and paired off sites averaged 1.0% (P = 0.060). When converted to a typical 14-day incubation period, predation rates averaged 14% higher on colonies (57.7 ?? 2.7%; ?? ?? SE) than at off sites (50.4 ?? 3.1%). Comparisons of habitat variables on colonies to off sites showed percent canopy cover of vegetation was similar (P = 0.114), percent bare ground was higher on colonies (P 0.288). Although we found the risk of nest predation was higher on white-tailed prairie dog colonies than at off sites, fitness of birds nesting on colonies might depend on other factors that influence foraging success, reproductive success, or nestling survival.

  13. Genetic diversity affects colony survivorship in commercial honey bee colonies

    NASA Astrophysics Data System (ADS)

    Tarpy, David R.; vanEngelsdorp, Dennis; Pettis, Jeffrey S.

    2013-08-01

    Honey bee ( Apis mellifera) queens mate with unusually high numbers of males (average of approximately 12 drones), although there is much variation among queens. One main consequence of such extreme polyandry is an increased diversity of worker genotypes within a colony, which has been shown empirically to confer significant adaptive advantages that result in higher colony productivity and survival. Moreover, honey bees are the primary insect pollinators used in modern commercial production agriculture, and their populations have been in decline worldwide. Here, we compare the mating frequencies of queens, and therefore, intracolony genetic diversity, in three commercial beekeeping operations to determine how they correlate with various measures of colony health and productivity, particularly the likelihood of queen supersedure and colony survival in functional, intensively managed beehives. We found the average effective paternity frequency ( m e ) of this population of honey bee queens to be 13.6 ± 6.76, which was not significantly different between colonies that superseded their queen and those that did not. However, colonies that were less genetically diverse (headed by queens with m e ≤ 7.0) were 2.86 times more likely to die by the end of the study when compared to colonies that were more genetically diverse (headed by queens with m e > 7.0). The stark contrast in colony survival based on increased genetic diversity suggests that there are important tangible benefits of increased queen mating number in managed honey bees, although the exact mechanism(s) that govern these benefits have not been fully elucidated.

  14. Genetic diversity affects colony survivorship in commercial honey bee colonies.

    PubMed

    Tarpy, David R; Vanengelsdorp, Dennis; Pettis, Jeffrey S

    2013-08-01

    Honey bee (Apis mellifera) queens mate with unusually high numbers of males (average of approximately 12 drones), although there is much variation among queens. One main consequence of such extreme polyandry is an increased diversity of worker genotypes within a colony, which has been shown empirically to confer significant adaptive advantages that result in higher colony productivity and survival. Moreover, honey bees are the primary insect pollinators used in modern commercial production agriculture, and their populations have been in decline worldwide. Here, we compare the mating frequencies of queens, and therefore, intracolony genetic diversity, in three commercial beekeeping operations to determine how they correlate with various measures of colony health and productivity, particularly the likelihood of queen supersedure and colony survival in functional, intensively managed beehives. We found the average effective paternity frequency (m e ) of this population of honey bee queens to be 13.6 ± 6.76, which was not significantly different between colonies that superseded their queen and those that did not. However, colonies that were less genetically diverse (headed by queens with m e  ≤ 7.0) were 2.86 times more likely to die by the end of the study when compared to colonies that were more genetically diverse (headed by queens with m e  > 7.0). The stark contrast in colony survival based on increased genetic diversity suggests that there are important tangible benefits of increased queen mating number in managed honey bees, although the exact mechanism(s) that govern these benefits have not been fully elucidated. PMID:23728203

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites. PMID:26786892

  19. Anti-Tuberculosis Policy of the Government General of Korea during Japanese-Colonial Period (1910-1945): From Simple Restriction to Active Enlightenment.

    PubMed

    Choi, Eun Kyung

    2013-12-01

    In this paper, I tried to examine the characteristic of anti-tuberculosis policy in colonial Korea and find out internal constraint of hygienic administration by Japanese government during Japanese-Colonial Period. Despite of high prevalence of tuberculosis among Japanese in Korea, the Japanese Government General of Korea had done almost nothing until 1936. Japan's hygienic administration was highly dependent upon hygienic police, and mainly with compulsory isolation and disinfection. It was inefficient in tuberculosis problem. In 1918, Japanese Government General enacted 'Ordinance of Prevention of Tuberculosis', solely based upon naive tuberculosis etiology in sputum; consisted of simple crackdown and isolation and had no effect due to the limit of anti-tuberculosis and health budget. Also the ordinance actually set limitation upon the tuberculosis facilities, only a few health care facilities could be affordable for tuberculosis patients. Since 1936, the Japanese Government General of Korea began tuberculosis prevention measures in earnest. Due to the Second Sino- Japanese War and World War II, there was urgent need to make Korean society and population as "safe, and healthy rear area". The Government organized 'Chosen Anti-tuberculosis Association' and highly pursued enlightment campaign. It was almost temporary measures of enlightenment and publicity. Also various types of health screening and tuberculosis prevalence research were introduced to Korean people. But it was not so effective to identify tuberculosis problem in Korea. Mass tuberculin test and X-ray test was introduced, but it was not well organized and scientifically designed. Besides, tuberculosis treatment facility was extremely rare because of strict isolation and high standard policy. Japanese Governemtn set numerous tuberculosis-counseling centers and mobilized public doctor for consulting tuberculosis, but the accessibility of centers was very low. Moreover, there was no source to establish

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

    SciTech Connect

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

    2008-11-10

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

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

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

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

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

    PubMed

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

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes. PMID:25449264

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

    SciTech Connect

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

    2014-01-03

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

  4. Characterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2013-12-01

    The goal of this project is to estimate volume loss of soil over time from this site, provide parameterizations on erodibility of ice rich permafrost and serve as a baseline for future landscape evolution simulations. Located in the zone of discontinuous permafrost, the interior region of Alaska (USA) is home to a large quantity of warm, unstable permafrost that is both high in ice content and has soil temperatures near the freezing point. Much of this permafrost maintains a frozen state despite the general warming air temperature trend in the region due to the presence of a thick insulating organic mat and a dense root network in the upper sub-surface of the soil column. At a rapidly evolving thermo-erosion site, located within the Caribou-Poker Creeks Research Watershed (part of the Bonanza Creek LTER) near Chatanika, Alaska (N65.140, W147.570), the protective organic layer and associated plants were disturbed by an adjacent traditional use trail and the shifting of a groundwater spring. These triggers have led to rapid geomorphological change on the landscape as the soil thaws and sediment is transported into the creek at the valley bottom. Since 2006 (approximately the time of initiation), the thermal erosion has grown to 170 meters length, 3 meters max depth, and 15 meters maximum width. This research combines several data sets: DGPS survey, imagery from an extremely low altitude pole-based remote sensing (3 to 5 meters above ground level), and imagery from an Unmanned Aerial System (UAS) at about 60m altitude.

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

    ERIC Educational Resources Information Center

    Pauls, John

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

  6. Comparative studies on growth and physiological responses of unicellular and colonial Microcystis aeruginosa to Acorus calamus.

    PubMed

    Zhang, S-H; Chang, J-J; Cao, J-Y; Yang, C-L

    2015-02-01

    In order to explore the growth inhibition and physiological responses of unicellular and colonial Microcystis aeruginosa during coexistence with Acorus calamus, algal densities, chlorophyll a contents, exopolysaccharide (EPS) concentrations, malondialdehyde (MDA) contents, catalase (CAT) activities, and peroxidase (POD) activities of the two algae strains were analyzed. Although the unicellular and colonial strains of M. aeruginosa were both inhibited by A. calamus, unicellular algae were more sensitive than the colonial algae. The measurement results for EPS, MDA, CAT, and POD showed that unicellular M. aeruginosa had higher levels of stress related damage than colonial strains when they were exposed to the same density of A. calamus, and the cellular defense system of colonial M. aeruginosa was stronger than that of unicellular M. aeruginosa. Natural blooms of Microcystis are typically composed of colonial forms of M. aeruginosa, therefore future efforts to control such blooms, possibly through the development of new algicides, should focus on the unique characteristics of colonial M. aeruginosa strains. PMID:25416545

  7. Granulocyte colony-stimulating factor receptor signalling via Janus kinase 2/signal transducer and activator of transcription 3 in ovarian cancer

    PubMed Central

    Kumar, J; Fraser, F W; Riley, C; Ahmed, N; McCulloch, D R; Ward, A C

    2014-01-01

    Background: Ovarian cancer remains a major cause of cancer mortality in women, with only limited understanding of disease aetiology at the molecular level. Granulocyte colony-stimulating factor (G-CSF) is a key regulator of both normal and emergency haematopoiesis, and is used clinically to aid haematopoietic recovery following ablative therapies for a variety of solid tumours including ovarian cancer. Methods: The expression of G-CSF and its receptor, G-CSFR, was examined in primary ovarian cancer samples and a panel of ovarian cancer cell lines, and the effects of G-CSF treatment on proliferation, migration and survival were determined. Results: G-CSFR was predominantly expressed in high-grade serous ovarian epithelial tumour samples and a subset of ovarian cancer cell lines. Stimulation of G-CSFR-expressing ovarian epithelial cancer cells with G-CSF led to increased migration and survival, including against chemotherapy-induced apoptosis. The effects of G-CSF were mediated by signalling via the downstream JAK2/STAT3 pathway. Conclusion: This study suggests that G-CSF has the potential to impact on ovarian cancer pathogenesis, and that G-CSFR expression status should be considered in determining appropriate therapy. PMID:24220695

  8. Indians and Southern Colonial Statutes

    ERIC Educational Resources Information Center

    Kawashima, Yasuhide

    1974-01-01

    Southern statutes, with their dual nature of uniformity and diversity, were doubtlessly an essential source of law for the examination of complex legal relations between American Indians and Anglo Americans in the colonial South. (FF)

  9. Reduction of urease activity by interaction with the flap covering the active site.

    PubMed

    Macomber, Lee; Minkara, Mona S; Hausinger, Robert P; Merz, Kenneth M

    2015-02-23

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes, and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  10. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  11. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  12. Small Molecule Active Site Directed Tools for Studying Human Caspases.

    PubMed

    Poreba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Rut, Wioletta; Salvesen, Guy S; Drag, Marcin

    2015-11-25

    Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes. PMID:26551511

  13. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

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

  14. [Yeast Communities of Formica aquilonia Colonies].

    PubMed

    Maksimova, A; Glushakova, A M; Kachalkin, A V; Chernov, I Yu; Panteleeva, S N; Reznikova, Zh I

    2016-01-01

    Yeast abundance and species diversity in the colonies of Formica aquilonia ants in birch-pine forbs forest, Novosibirsk oblast, Russia, was studied. The average yeast number in the anthill material was 10³-10⁴CFU/g, reaching 10⁵ CFU/g in the hatching chambers. Typical litter species (Trichosporon monilfiforme and Cystofilobasidium capitatum) were predominant in soil and litter around the anthills. Apart from these species, ascomycete species of the family Debaryomycetaceae, Debaryomyces hansenii and Schwanniomyces vanrijiae, were predominant in the anthill material. Yeast population of the ants consisted exclusively of the members of these two species. Thus, highly specific yeast communities formed in the colonies of Formica aquilonia ants differ from the communities of surrounding soil. These differences are an instance of environment-forming activity of the ants. PMID:27301134

  15. School Pharmacist/School Environmental Hygienic Activities at School Site.

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs". PMID:27252053

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

    PubMed Central

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

    2015-01-01

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

  17. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability. PMID:25671686

  18. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  19. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  20. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

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

    EPA Science Inventory

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

  2. Heritable choice of colony size in cliff swallows: does experience trump genetics in older birds?

    PubMed

    Roche, Erin A; Brown, Charles R; Brown, Mary Bomberger

    2011-12-01

    The variation in breeding-colony size seen in populations of most colonial birds may reflect heritable choices made by individuals who are phenotypically specialized for particular social environments. Although a few studies have reported evidence for genetically based choice of group sizes in birds, we know relatively little about the extent to which animals potentially rely on experience versus innate preferences in deciding with how many conspecifics to settle at different times of their lives. We conducted a cross-fostering experiment in 1997-1998 on cliff swallows (Petrochelidon pyrrhonota) in southwestern Nebraska, USA, in which some individuals were reared in colonies different in size from those in which they were born. Breeding-colony sizes chosen by this cohort of birds were monitored by mark-recapture throughout their lives. A multistate mark-recapture analysis revealed that birds in their first breeding year chose colony sizes similar to those of their birth, regardless of their rearing environment, confirming a previous analysis. Beyond the first breeding year, however, cliff swallows' colony choice was less dependent on where they were born. Birds born in small colonies and reared in large colonies showed evidence of a delayed rearing effect, with these birds overwhelmingly choosing large colonies in later years. Heritabilities suggested strong genetic effects on first-year colony choice but not in later years. Cliff swallows' genetically based colony-size preferences their first year could be a way to ensure matching of their phenotype to an appropriate social environment as yearlings. In later years, familiarity with particular colony sites and available information on site quality may override innate group-size preferences when birds choose colonies. PMID:22247565

  3. Heritable choice of colony size in cliff swallows: does experience trump genetics in older birds?

    PubMed Central

    Roche, Erin A.; Brown, Charles R.; Brown, Mary Bomberger

    2011-01-01

    The variation in breeding-colony size seen in populations of most colonial birds may reflect heritable choices made by individuals who are phenotypically specialized for particular social environments. Although a few studies have reported evidence for genetically based choice of group sizes in birds, we know relatively little about the extent to which animals potentially rely on experience versus innate preferences in deciding with how many conspecifics to settle at different times of their lives. We conducted a cross-fostering experiment in 1997–1998 on cliff swallows (Petrochelidon pyrrhonota) in southwestern Nebraska, USA, in which some individuals were reared in colonies different in size from those in which they were born. Breeding-colony sizes chosen by this cohort of birds were monitored by mark-recapture throughout their lives. A multistate mark-recapture analysis revealed that birds in their first breeding year chose colony sizes similar to those of their birth, regardless of their rearing environment, confirming a previous analysis. Beyond the first breeding year, however, cliff swallows’ colony choice was less dependent on where they were born. Birds born in small colonies and reared in large colonies showed evidence of a delayed rearing effect, with these birds overwhelmingly choosing large colonies in later years. Heritabilities suggested strong genetic effects on first-year colony choice but not in later years. Cliff swallows’ genetically based colony-size preferences their first year could be a way to ensure matching of their phenotype to an appropriate social environment as yearlings. In later years, familiarity with particular colony sites and available information on site quality may override innate group-size preferences when birds choose colonies. PMID:22247565

  4. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

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

  5. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  6. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-05

    ...BOEM is reopening the comment period announced in the Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and Site Assessment Activities on the OCS Offshore North...

  7. The relevance of the Mediterranean Region to colonial waterbird conservation

    USGS Publications Warehouse

    Erwin, R.M.

    1996-01-01

    The Mediterranean Sea is the largest partially enclosed sea in the world and provides habitat to more than 100 species of waterbirds from the Palearctic-North African-Middle Eastern regions. Even though the Mediterranean suffers from pollution, has little tidal influence, and is oligotrophic, more than half of the western Palearctic populations of numerous waterfowl species winter in the region. Thirty-three species of colonial waterbirds breed along the 46,000 km Mediterranean coastline with nine species considered threatened or endangered, mostly because of wetland loss and degradation. The long history of human activity and scientific investigations in the region has taught some valuable lessons. In the area of colonial waterbird biology and conservation, we have learned important lessons about the value of long-term monitoring and research on selected populations. From marking studies of Greater Flamingos (Phoenicopterus ruber roseus) and Little Egrets (Egretta garzetta) results have been used to derive useful information about metapopulation dynamics. Involvement of both African and European biologists allowed year-round Studies of these species that yielded valuable spin-offs for training in avian and wetland conservation. We have also learned the value of man-made wetlands as feeding and nesting sites for some colonial waterbirds. Careful evaluations of the habitat quality of different types of wetlands are required, as in contaminant levels such as lead shot and pesticides. Wetland conservationists have also learned from some instructive mistakes. Dam construction and agricultural incentive programs sponsored by the European Community, the World Bank, and others from the past have largely ignored impacts on wetlands and wildlife. In some areas, economic ventures such as aquaculture operations and salt mining have not involved waterbird habitat needs in their planning. Research and conservation needs include: (1) establishing regional monitoring programs and

  8. Measurement of ammonia emissions from tropical seabird colonies

    NASA Astrophysics Data System (ADS)

    Riddick, S. N.; Blackall, T. D.; Dragosits, U.; Daunt, F.; Braban, C. F.; Tang, Y. S.; MacFarlane, W.; Taylor, S.; Wanless, S.; Sutton, M. A.

    2014-06-01

    The excreta (guano) of seabirds at their breeding colonies represents a notable source of ammonia (NH3) emission to the atmosphere, with effects on surrounding ecosystems through nitrogen compounds being thereby transported from sea to land. Previous measurements in temperate UK conditions quantified emission hotspots and allowed preliminary global upscaling. However, thermodynamic processes and water availability limit NH3 formation from guano, which suggests that the proportion of excreted nitrogen that volatilizes as NH3 may potentially be higher at tropical seabird colonies than similar colonies in temperate or sub-polar regions. To investigate such differences, we measured NH3 concentrations and environmental conditions at two tropical seabird colonies during the breeding season: a colony of 20,000 tern spp. and noddies on Michaelmas Cay, Great Barrier Reef, and a colony of 200,000 Sooty terns on Ascension Island, Atlantic Ocean. At both sites time-integrated NH3 concentrations and meteorological parameters were measured. In addition, at Ascension Island, semi-continuous hourly NH3 concentrations and micrometeorological parameters were measured throughout the campaign. Ammonia emissions, quantified using a backwards Lagrangian atmospheric dispersion model, were estimated at 21.8 μg m-2 s-1 and 18.9 μg m-2 s-1 from Michaelmas Cay and Ascension Island, respectively. High temporal resolution NH3 data at Ascension Island estimated peak hourly emissions up to 377 μg NH3 m2 s-1. The estimated percentage fraction of total guano nitrogen volatilized was 67% at Michaelmas Cay and 32% at Ascension Island, with the larger value at the former site attributed to higher water availability. These values are much larger than published data for sub-polar locations, pointing to a substantial climatic dependence on emission of atmospheric NH3 from seabird colonies.

  9. Zimbabwe Colonial and Post-Colonial Language Policy and Planning Practices

    ERIC Educational Resources Information Center

    Makoni, Sinfree B.; Dube, Busi; Mashiri, Pedzisai

    2006-01-01

    This monograph focuses on the development of colonial and post-colonial language policies and practices in Zimbabwe, attributing changes to evolving philosophies and politics in colonial and post-colonial Zimbabwe. In colonial Zimbabwe, we argue that the language policies had as one of their key objectives the development of a bilingual white…

  10. Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

    PubMed Central

    2015-01-01

    Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

  11. The Role of Non-Foraging Nests in Polydomous Wood Ant Colonies

    PubMed Central

    Ellis, Samuel; Robinson, Elva J. H.

    2015-01-01

    A colony of red wood ants can inhabit more than one spatially separated nest, in a strategy called polydomy. Some nests within these polydomous colonies have no foraging trails to aphid colonies in the canopy. In this study we identify and investigate the possible roles of non-foraging nests in polydomous colonies of the wood ant Formica lugubris. To investigate the role of non-foraging nests we: (i) monitored colonies for three years; (ii) observed the resources being transported between non-foraging nests and the rest of the colony; (iii) measured the amount of extra-nest activity around non-foraging and foraging nests. We used these datasets to investigate the extent to which non-foraging nests within polydomous colonies are acting as: part of the colony expansion process; hunting and scavenging specialists; brood-development specialists; seasonal foragers; or a selfish strategy exploiting the foraging effort of the rest of the colony. We found that, rather than having a specialised role, non-foraging nests are part of the process of colony expansion. Polydomous colonies expand by founding new nests in the area surrounding the existing nests. Nests founded near food begin foraging and become part of the colony; other nests are not founded near food sources and do not initially forage. Some of these non-foraging nests eventually begin foraging; others do not and are abandoned. This is a method of colony growth not available to colonies inhabiting a single nest, and may be an important advantage of the polydomous nesting strategy, allowing the colony to expand into profitable areas. PMID:26465750

  12. The Role of Non-Foraging Nests in Polydomous Wood Ant Colonies.

    PubMed

    Ellis, Samuel; Robinson, Elva J H

    2015-01-01

    A colony of red wood ants can inhabit more than one spatially separated nest, in a strategy called polydomy. Some nests within these polydomous colonies have no foraging trails to aphid colonies in the canopy. In this study we identify and investigate the possible roles of non-foraging nests in polydomous colonies of the wood ant Formica lugubris. To investigate the role of non-foraging nests we: (i) monitored colonies for three years; (ii) observed the resources being transported between non-foraging nests and the rest of the colony; (iii) measured the amount of extra-nest activity around non-foraging and foraging nests. We used these datasets to investigate the extent to which non-foraging nests within polydomous colonies are acting as: part of the colony expansion process; hunting and scavenging specialists; brood-development specialists; seasonal foragers; or a selfish strategy exploiting the foraging effort of the rest of the colony. We found that, rather than having a specialised role, non-foraging nests are part of the process of colony expansion. Polydomous colonies expand by founding new nests in the area surrounding the existing nests. Nests founded near food begin foraging and become part of the colony; other nests are not founded near food sources and do not initially forage. Some of these non-foraging nests eventually begin foraging; others do not and are abandoned. This is a method of colony growth not available to colonies inhabiting a single nest, and may be an important advantage of the polydomous nesting strategy, allowing the colony to expand into profitable areas. PMID:26465750

  13. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

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

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

  14. Protein-ligand docking using fitness learning-based artificial bee colony with proximity stimuli.

    PubMed

    Uehara, Shota; Fujimoto, Kazuhiro J; Tanaka, Shigenori

    2015-07-01

    Protein-ligand docking is an optimization problem, which aims to identify the binding pose of a ligand with the lowest energy in the active site of a target protein. In this study, we employed a novel optimization algorithm called fitness learning-based artificial bee colony with proximity stimuli (FlABCps) for docking. Simulation results revealed that FlABCps improved the success rate of docking, compared to four state-of-the-art algorithms. The present results also showed superior docking performance of FlABCps, in particular for dealing with highly flexible ligands and proteins with a wide and shallow binding pocket. PMID:26050878

  15. Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

    PubMed Central

    Seibold, Steve A.; Singh, Badri Nath; Zhang, Chunfen; Kireeva, Maria; Domecq, Céline; Bouchard, Annie; Nazione, Anthony M.; Feig, Michael; Cukier, Robert I.; Coulombe, Benoit; Kashlev, Mikhail; Hampsey, Michael; Burton, Zachary F.

    2010-01-01

    Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed. PMID:20478425

  16. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins. PMID:24635441

  17. Active sites for NO reduction over Fe-ZSM-5 catalysts.

    PubMed

    Schwidder, M; Santhosh Kumar, M; Brückner, A; Grünert, W

    2005-02-14

    A study of Fe-ZSM-5 catalysts with variable amounts of isolated, oligomeric and heavily aggregated Fe3+ oxo sites (as evidenced by UV-Vis and EPR spectroscopic data) and their catalytic properties in the selective catalytic reduction of NO by isobutane or by NH3 is presented, which allows development of a unified concept of the active Fe sites in these reactions, according to which isolated Fe sites catalyse both SCR reactions while oligomeric sites, though also involved in the selective reduction path, limit the catalyst performance by causing the total oxidation of the reductant. PMID:15685345

  18. Responses to human intruders by birds nesting in colonies: Experimental results and management guidelines

    USGS Publications Warehouse

    Erwin, R.M.

    1989-01-01

    Colonies of nesting wading birds and seabirds were studied at coastal sites in Virginia and North Carolina to determine distances at which birds flushed in response to human intrusion. There were few statistically significant relationships between flushing distances and colony size. Similarly, there were few differences between responses during incubation compared to post-hatching periods.

  19. Differential regulation of early response genes and cell proliferation through the human granulocyte macrophage colony-stimulating factor receptor: selective activation of the c-fos promoter by genistein.

    PubMed Central

    Watanabe, S; Muto, A; Yokota, T; Miyajima, A; Arai, K

    1993-01-01

    Granulocyte macrophage colony-stimulating factor (GM-CSF) binds to the high-affinity GM-CSF receptor (GMR) consisting of alpha and beta subunits and induces rapid tyrosine phosphorylation, activation of early response genes, and proliferation of hematopoietic cells. The alpha subunit is the primary cytokine binding component and the beta subunit is required for high-affinity binding as well as for signal transduction. Using tyrosine kinase inhibitors and cytoplasmic deletion mutants of the beta subunit, we obtained evidence that there are at least two distinct pathways downstream of the GMR in BA/F3 cell, one which is essential for proliferation, leads to the c-myc gene activation, and is sensitive to herbimycin and genistein. Activation of this pathway depends on the cytoplasmic region between amino acid positions 455 and 517 of the beta subunit. The second pathway, which leads to activation of c-fos and c-jun genes, is only partially sensitive to herbimycin, is resistant to genistein and depends on the region between amino acid positions 626 and 763 of the beta subunit. Unexpectedly, the c-fos mRNA induction was augmented by genistein. The enhanced expression of c-fos mRNA by genistein also occurred with stimulation with cAMP, PMA, or EGF in NIH3T3 cells. It thus seems likely that genistein affects a common pathway downstream of these signals. Images PMID:8298195

  20. Colony-stimulating factor-1 (CSF-1) receptor-mediated macrophage differentiation in myeloid cells: a role for tyrosine 559-dependent protein phosphatase 2A (PP2A) activity.

    PubMed Central

    McMahon, K A; Wilson, N J; Marks, D C; Beecroft, T L; Whitty, G A; Hamilton, J A; Csar, X F

    2001-01-01

    M1 myeloid cells transfected with the wild-type (WT) colony-stimulating factor-1 (CSF-1) receptor (CSF-1R; M1/WT cells) undergo CSF-1-dependent macrophage differentiation. By mutation studies, we have provided prior evidence that tyrosine 559 in the CSF-1R cytoplasmic domain governs the Src-dependent differentiation pathway. Further components of this pathway were then sought. We report that the extent of CSF-1-mediated tyrosine phosphorylation of protein phosphatase 2A (PP2A), and the associated loss of its activity were reduced in M1 cells transfected with the CSF-1R with a tyrosine-to-phenylalanine mutation at position 559 (M1/559 cells), compared with the corresponding responses in CSF-1-treated M1/WT cells. This evidence for an involvement of a reduction in PP2A activity in the differentiation process was supported by the restoration of the defect in the CSF-1-mediated differentiation of M1/559 cells by the addition of the PP2A inhibitor, okadaic acid. It was also found that the degree of activation of extracellular-signal-regulated kinase (ERK) activities by CSF-1 was reduced in M1/559 cells, suggesting their involvement in the differentiation process. These data suggest that PP2A and ERK form part of the Src-dependent signal-transduction cascade governing CSF-1-mediated macrophage differentiation in M1 cells. PMID:11513742

  1. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  2. Redox signaling in the growth and development of colonial hydroids.

    PubMed

    Blackstone, Neil W

    2003-02-01

    Redox signaling provides a quick and efficient mechanism for clonal or colonial organisms to adapt their growth and development to aspects of the environment, e.g. the food supply. A 'signature' of mitochondrial redox signaling, particularly as mediated by reactive oxygen species (ROS), can be elucidated by experimental manipulation of the electron transport chain. The major sites of ROS formation are found at NADH dehydrogenase of complex I and at the interface between coenzyme Q and complex III. Inhibitors of complex III should thus upregulate ROS from both sites; inhibitors of complex I should upregulate ROS from the first but not the second site, while uncouplers of oxidative phosphorylation should downregulate ROS from both sites. To investigate the possibility of such redox signaling, perturbations of colony growth and development were carried out using the hydroid Podocoryna carnea. Oxygen uptake of colonies was measured to determine comparable physiological doses of antimycin A(1) (an inhibitor of complex III), rotenone (an inhibitor of complex I) and carbonyl cyanide m-chlorophenylhydrazone (CCCP; an uncoupler of oxidative phosphorylation). Using these doses, clear effects on colony growth and development were obtained. Treatment with antimycin A(1) results in 'runner-like' colony growth, with widely spaced polyps and stolon branches, while treatment with CCCP results in 'sheet-like' growth, with closely spaced polyps and stolon branches. Parallel results have been obtained previously with azide, an inhibitor of complex IV, and dinitrophenol, another uncoupler of oxidative phosphorylation. Perhaps surprisingly, rotenone produced effects on colony development similar to those of CCCP. Assays of peroxides using 2',7'-dichlorofluorescin diacetate and fluorescent microscopy suggest a moderate difference in ROS formation between the antimycin and rotenone treatments. The second site of ROS formation (the interface between coenzyme Q and complex III) may thus

  3. Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis.

    PubMed Central

    Gosert, R; Dollenmaier, G; Weitz, M

    1997-01-01

    Picornavirus 3C proteases (3Cpro) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3Cpro of hepatitis A virus (HAV) to those of other picornaviruses have resulted in prediction of active-site residues: histidine at position 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonucleotide-directed mutagenesis was targeted to 3Cpro in the context of natural polypeptide precursor P3. Autocatalytic processing of the polyprotein containing wild-type or variant 3Cpro was tested by in vivo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybrid system and by in vitro translation of corresponding RNAs. Comparison with proteins present in HAV-infected cells showed that both expression systems mimicked authentic polyprotein processing. Individual substitutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contrast, a P3 polyprotein in which D98 was substituted by asparagine underwent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more pronounced loss of processing. Therefore, apparently H44 and C172 are active-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprotein cleavage sites may reduce proteolytic activity, presumably by altering substrate conformation. PMID:9060667

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

    PubMed Central

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

    2012-01-01

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site. PMID:21926991

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

    SciTech Connect

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

    2011-09-18

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

  6. Effect of formic acid formulations on honey bee (Hymenoptera: Apidae) colonies and influence of colony and ambient conditions on formic acid concentration in the hive.

    PubMed

    Ostermann, David J; Currie, Robert W

    2004-10-01

    The interaction between the effects of varroa, Varroa destructor Anderson & Trueman, and formic acid treatments on colonies of honey bees, Apis mellifera L., were examined in two field experiments. In experiment 1, colonies with low varroa levels were exposed to two different slow-release formulations and compared with untreated colonies. In experiment 2, colonies inoculated with varroa and uninoculated colonies were exposed to a slow-release formulation, a pour-on formulation, or were left untreated. The effects of treatments, hive temperature, and hive relative humidity on formic acid concentration in hive air also were examined. Slow-release formic acid application improved colony development in colonies that had been inoculated with varroa. However, in uninoculated colonies where the mean abundance of varroa was low, slow-release formic acid application suppressed colony development. The pour-on application did not have a negative impact on worker population growth in uninoculated colonies, but also it was not as effective as the slow-release treatment in improving population growth in varroa-inoculated colonies. Equivalent volumes of acid applied in pour-on and slow-release formulations provided the same cumulative dose in hive air but differed in the daily pattern of formic acid release. Colonies that were not inoculated with varroa had higher concentrations of formic acid in hive air than colonies that were inoculated with varroa on three of the five pour-on application dates. The data suggest that reductions in worker population and/or activity caused by varroa can interact with ambient conditions to affect the volatilization or sorption of formic acid in the hive. PMID:15568335

  7. HIV integration site distributions in resting and activated CD4+ T cells infected in culture

    PubMed Central

    Brady, Troy; Agosto, Luis M.; Malani, Nirav; Berry, Charles C.; O'Doherty, Una; Bushman, Frederic

    2010-01-01

    Objective The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting. Design Primary resting or activated CD4+ T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined. Methods We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions. Results HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes. Conclusion These data indicate that integration site distributions show modest but significant differences between resting and activated CD4+ T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression. PMID:19550285

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

    SciTech Connect

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

    2007-01-01

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

  9. Assessment of the site of ventricular activation by Fourier analysis of gated blood-pool studies

    SciTech Connect

    Links, J.M.; Raichlen, J.S.; Wagner, H.N. Jr.; Reid, P.R.

    1985-01-01

    The authors studied the use of first-harmonic Fourier analysis of gated blood-pool images to assess the site of ventricular activation in a group of 12 patients undergoing electrophysiologic pacing studies. They acquired gated blood-pool studies during pacing at up to four sites at each of two different rates. A total of 50 studies were made. At a pacing rate of 100 beats/min, when the pacing electrode was the right-ventricular outflow tract, 7/8; at the anterolateral left-ventricular wall, 4/4. When the Fourier activation site was at the right-ventricular apex, 9/9 times the pacing electrode was there; at the right-ventricular outflow tract, 7/10; in the left ventricle, 4/4. Fourier analysis of gated blood-pool studies can help identify the site of ventricular activation but is not sufficiently accurate to fully replace endocardial mapping.

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

    SciTech Connect

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

    2009-01-01

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

  11. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

    Gould, Christine M.; Antal, Corina E.; Reyes, Gloria; Kunkel, Maya T.; Adams, Ryan A.; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C.

    2011-01-01

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C. PMID:21715334

  12. Chronic sublethal stress causes bee colony failure

    PubMed Central

    Bryden, John; Gill, Richard J; Mitton, Robert A A; Raine, Nigel E; Jansen, Vincent A A; Hodgson, David

    2013-01-01

    Current bee population declines and colony failures are well documented yet poorly understood and no single factor has been identified as a leading cause. The evidence is equivocal and puzzling: for instance, many pathogens and parasites can be found in both failing and surviving colonies and field pesticide exposure is typically sublethal. Here, we investigate how these results can be due to sublethal stress impairing colony function. We mathematically modelled stress on individual bees which impairs colony function and found how positive density dependence can cause multiple dynamic outcomes: some colonies fail while others thrive. We then exposed bumblebee colonies to sublethal levels of a neonicotinoid pesticide. The dynamics of colony failure, which we observed, were most accurately described by our model. We argue that our model can explain the enigmatic aspects of bee colony failures, highlighting an important role for sublethal stress in colony declines. PMID:24112478

  13. Genetic diversity affects colony survivorship in commercial honey bee colonies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Honey bee (Apis mellifera) queens mate with unusually high numbers of males (average of approximately 12 drones), although there is much variation among queens. One main consequence of such extreme polyandry is an increased diversity of worker genotypes within a colony, which has been shown empirica...

  14. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents.

    PubMed

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-04-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, a(w). The apparent turnover number k(cat)(app) in these systems ranged from 0.2 to 80 s(-1) and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing a(w). (19)F NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on a(w). The rates of NMR-derived fast (k > 10(7) s(-1)) and slow (k < 10(4) s(-1)) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with k(cat). The (19)F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant epsilon(as), which increased with hydration to epsilon(as) approximately 13 in each solvent. In both solvents, the transition state free energy data and epsilon(as) followed Kirkwood's model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site's electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  15. Effects of resource activities upon repository siting and waste containment with reference to bedded salt

    SciTech Connect

    Ashby, J.; Rowe, J.

    1980-02-01

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases.

  16. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    PubMed

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO. PMID:15901543

  20. Glucose induced fractal colony pattern of Bacillus thuringiensis.

    PubMed

    Roy, Manas K; Banerjee, Paromita; Sengupta, Tapas K; Dattagupta, Sushanta

    2010-08-01

    Growing colonies of bacteria on the surface of thin agar plates exhibit fractal patterns as a result of nonlinear response to environmental conditions, such as nutrients, solidity of the agar medium and temperature. Here, we examine the effect of glucose on pattern formation by growing colonies of Bacillus thuringiensis isolate KPWP1. We also present the theoretical modeling of the colony growth of KPWP1 and the associated spatio-temporal patterns. Our experimental results are in excellent agreement with simulations based on a reaction-diffusion model that describes diffusion-limited aggregation and branching, in which individual cells move actively in the periphery, but become immotile in the inner regions of the growing colony. We obtain the Hausdorff fractal dimension of the colony patterns: D(H.Expt)=1.1969 and D(H, R.D.=)1.1965, for experiment and reaction-diffusion model, respectively. Results of our experiments and modeling clearly show how glucose at higher concentration can prove to be inhibitory for motility of growing colonies of B. thuringiensis cells on semisolid support and be responsible for changes in the growth pattern. PMID:20553734

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

    PubMed Central

    Ping, Z A; Butterfiel, D A

    1991-01-01

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

  2. Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

    PubMed

    Ozeri-Galai, Efrat; Lebofsky, Ronald; Rahat, Ayelet; Bester, Assaf C; Bensimon, Aaron; Kerem, Batsheva

    2011-07-01

    Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development. PMID:21726815

  3. Active-site mobility revealed by the crystal structure of arylmalonate decarboxylase from Bordetella bronchiseptica.

    PubMed

    Kuettner, E Bartholomeus; Keim, Antje; Kircher, Markus; Rosmus, Susann; Sträter, Norbert

    2008-03-21

    Arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of arylmethylmalonates without the need for an organic cofactor or metal ion. The decarboxylation reaction is of interest for the synthesis of fine chemicals. As basis for an analysis of the catalytic mechanism of AMDase and for a rational enzyme design, we determined the X-ray structure of the enzyme up to 1.9 A resolution. Like the distantly related aspartate or glutamate racemases, AMDase has an aspartate transcarbamoylase fold consisting of two alpha/beta domains related by a pseudo dyad. However, the domain orientation of AMDase differs by about 30 degrees from that of the glutamate racemases, and also significant differences in active-site structures are observed. In the crystals, four independent subunits showing different conformations of active-site loops are present. This finding is likely to reflect the active-site mobility necessary for catalytic activity. PMID:18258259

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

    SciTech Connect

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

    2014-12-12

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

  5. 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. PMID:27285815

  6. Parent–offspring resemblance in colony-specific adult survival of cliff swallows

    USGS Publications Warehouse

    Brown, Charles R.; Roche, Erin A.; Brown, Mary Bomberger

    2015-01-01

    Survival is a key component of fitness. Species that occupy discrete breeding colonies with different characteristics are often exposed to varying costs and benefits associated with group size or environmental conditions, and survival is an integrative net measure of these effects. We investigated the extent to which survival probability of adult (≥1-year old) cliff swallows (Petrochelidon pyrrhonota) occupying different colonies resembled that of their parental cohort and thus whether the natal colony had long-term effects on individuals. Individuals were cross-fostered between colonies soon after hatching and their presence as breeders monitored at colonies in the western Nebraska study area for the subsequent decade. Colony-specific adult survival probabilities of offspring born and reared in the same colony, and those cross-fostered away from their natal colony soon after birth, were positively and significantly related to subsequent adult survival of the parental cohort from the natal colony. This result held when controlling for the effect of natal colony size and the age composition of the parental cohort. In contrast, colony-specific adult survival of offspring cross-fostered to a site was unrelated to that of their foster parent cohort or to the cohort of non-fostered offspring with whom they were reared. Adult survival at a colony varied inversely with fecundity, as measured by mean brood size, providing evidence for a survival–fecundity trade-off in this species. The results suggest some heritable variation in adult survival, likely maintained by negative correlations between fitness components. The study provides additional evidence that colonies represent non-random collections of individuals.

  7. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

    PubMed Central

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

  8. Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues.

    PubMed

    Jia, Y; Lu, Z; Huang, K; Herzberg, O; Dunaway-Mariano, D

    1999-10-26

    PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed. PMID:10571990

  9. Localization of the binding site of tissue-type plasminogen activator to fibrin.

    PubMed Central

    Ichinose, A; Takio, K; Fujikawa, K

    1986-01-01

    Functionally active A and B chains were separated from a two-chain form of recombinant tissue-type plasminogen activator after mild reduction and alkylation. The A chain was found to be responsible for the binding to lysine-Sepharose or fibrin and the B chain contained the catalytic activity of tissue-type plasminogen activator. An extensive reduction of two-chain tissue-type plasminogen activator, however, destroyed both the binding and catalytic activities. A thermolytic fragment, Fr. 1, of tissue-type plasminogen activator that contained a growth factor and two kringle segments retained its lysine binding activity. Additional thermolytic cleavages in the kringle-2 segment of Fr. 1 caused a total loss of the binding activity. These results indicated that the binding site of tissue-type plasminogen activator to fibrin was located in the kringle-2 segment. Images PMID:3088041

  10. Variation in Symbiodinium ITS2 sequence assemblages among coral colonies.

    PubMed

    Stat, Michael; Bird, Christopher E; Pochon, Xavier; Chasqui, Luis; Chauka, Leonard J; Concepcion, Gregory T; Logan, Dan; Takabayashi, Misaki; Toonen, Robert J; Gates, Ruth D

    2011-01-01

    Endosymbiotic dinoflagellates in the genus Symbiodinium are fundamentally important to the biology of scleractinian corals, as well as to a variety of other marine organisms. The genus Symbiodinium is genetically and functionally diverse and the taxonomic nature of the union between Symbiodinium and corals is implicated as a key trait determining the environmental tolerance of the symbiosis. Surprisingly, the question of how Symbiodinium diversity partitions within a species across spatial scales of meters to kilometers has received little attention, but is important to understanding the intrinsic biological scope of a given coral population and adaptations to the local environment. Here we address this gap by describing the Symbiodinium ITS2 sequence assemblages recovered from colonies of the reef building coral Montipora capitata sampled across Kāne'ohe Bay, Hawai'i. A total of 52 corals were sampled in a nested design of Coral Colony(Site(Region)) reflecting spatial scales of meters to kilometers. A diversity of Symbiodinium ITS2 sequences was recovered with the majority of variance partitioning at the level of the Coral Colony. To confirm this result, the Symbiodinium ITS2 sequence diversity in six M. capitata colonies were analyzed in much greater depth with 35 to 55 clones per colony. The ITS2 sequences and quantitative composition recovered from these colonies varied significantly, indicating that each coral hosted a different assemblage of Symbiodinium. The diversity of Symbiodinium ITS2 sequence assemblages retrieved from individual colonies of M. capitata here highlights the problems inherent in interpreting multi-copy and intra-genomically variable molecular markers, and serves as a context for discussing the utility and biological relevance of assigning species names based on Symbiodinium ITS2 genotyping. PMID:21246044

  11. Variation in Symbiodinium ITS2 Sequence Assemblages among Coral Colonies

    PubMed Central

    Stat, Michael; Bird, Christopher E.; Pochon, Xavier; Chasqui, Luis; Chauka, Leonard J.; Concepcion, Gregory T.; Logan, Dan; Takabayashi, Misaki; Toonen, Robert J.; Gates, Ruth D.

    2011-01-01

    Endosymbiotic dinoflagellates in the genus Symbiodinium are fundamentally important to the biology of scleractinian corals, as well as to a variety of other marine organisms. The genus Symbiodinium is genetically and functionally diverse and the taxonomic nature of the union between Symbiodinium and corals is implicated as a key trait determining the environmental tolerance of the symbiosis. Surprisingly, the question of how Symbiodinium diversity partitions within a species across spatial scales of meters to kilometers has received little attention, but is important to understanding the intrinsic biological scope of a given coral population and adaptations to the local environment. Here we address this gap by describing the Symbiodinium ITS2 sequence assemblages recovered from colonies of the reef building coral Montipora capitata sampled across Kāne'ohe Bay, Hawai'i. A total of 52 corals were sampled in a nested design of Coral Colony(Site(Region)) reflecting spatial scales of meters to kilometers. A diversity of Symbiodinium ITS2 sequences was recovered with the majority of variance partitioning at the level of the Coral Colony. To confirm this result, the Symbiodinium ITS2 sequence diversity in six M. capitata colonies were analyzed in much greater depth with 35 to 55 clones per colony. The ITS2 sequences and quantitative composition recovered from these colonies varied significantly, indicating that each coral hosted a different assemblage of Symbiodinium. The diversity of Symbiodinium ITS2 sequence assemblages retrieved from individual colonies of M. capitata here highlights the problems inherent in interpreting multi-copy and intra-genomically variable molecular markers, and serves as a context for discussing the utility and biological relevance of assigning species names based on Symbiodinium ITS2 genotyping. PMID:21246044

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

    SciTech Connect

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

    2009-11-10

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

  13. Effects of a Fipronil Spot-Treatment on field colonies of Coptotermes formosanus (Isoptera: Rhinotermitidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This field study investigated the colony effect of a Fipronil spot-treatment applied to active infestations of Formosan subterranean termite, Coptotermes formosanus Shiraki. Spot-treatments were applied to a single active independent monitor from each of four colonies in which multiple independent m...

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

    PubMed

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

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds. PMID:21877154

  15. Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

    PubMed Central

    Rinehart, Jesse; Maksimova, Yelena D.; Tanis, Jessica E.; Stone, Kathryn L.; Hodson, Caleb A.; Zhang, Junhui; Risinger, Mary; Pan, Weijun; Wu, Dianqing; Colangelo, Christopher M.; Forbush, Biff; Joiner, Clinton H.; Gulcicek, Erol E.; Gallagher, Patrick G.; Lifton, Richard P.

    2010-01-01

    Summary Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function. PMID:19665974

  16. The Vine and Olive Colony.

    ERIC Educational Resources Information Center

    Albinski, Nan Bowman

    1985-01-01

    Traces the historical sources of "Some Plant Olive Trees," a utopian novel by Emma Gelders Sterne, which offers a fictional account of the Vine and Olive colony, one of the most colorful yet least known utopian communities of the nineteenth century. (AYC)

  17. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO

    NASA Astrophysics Data System (ADS)

    Alfonso, Dominic R.; Kauffman, Douglas; Matranga, Christopher

    2016-05-01

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25- nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts.

  18. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO.

    PubMed

    Alfonso, Dominic R; Kauffman, Douglas; Matranga, Christopher

    2016-05-14

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25 (-) nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts. PMID:27179498

  19. Effects of introducing foxes and raccoons on herring gull colonies

    USGS Publications Warehouse

    Kadlec, J.A.

    1971-01-01

    Red foxes (Vulpes fulva) and raccoons (Procyon lotor) released at colonies of herring gulls (Larus argentatus) on islands off the Massachusetts coast effectively eliminated the production of young gulls. Annual predator introductions for 2-4 years caused major reductions in colony size and occasionally total abandonment of the island as a colony site. Observations of the experimental islands for 2 years after cessation of predator introductions showed slow repopulation of the islands and lower breeding success than on control islands. The size of the regional population was reduced largely because of the movements of gulls off the experimental islands. The introduced predators are, in most cases, difficult to maintain on the islands; this restricts their utility in population management.

  20. Estimating rates of local extinction and colonization in colonial species and an extension to the metapopulation and community levels

    USGS Publications Warehouse

    Barbraud, C.; Nichols, J.D.; Hines, J.E.; Hafner, H.

    2003-01-01

    Coloniality has mainly been studied from an evolutionary perspective, but relatively few studies have developed methods for modelling colony dynamics. Changes in number of colonies over time provide a useful tool for predicting and evaluating the responses of colonial species to management and to environmental disturbance. Probabilistic Markov process models have been recently used to estimate colony site dynamics using presence-absence data when all colonies are detected in sampling efforts. Here, we define and develop two general approaches for the modelling and analysis of colony dynamics for sampling situations in which all colonies are, and are not, detected. For both approaches, we develop a general probabilistic model for the data and then constrain model parameters based on various hypotheses about colony dynamics. We use Akaike's Information Criterion (AIC) to assess the adequacy of the constrained models. The models are parameterised with conditional probabilities of local colony site extinction and colonization. Presence-absence data arising from Pollock's robust capture-recapture design provide the basis for obtaining unbiased estimates of extinction, colonization, and detection probabilities when not all colonies are detected. This second approach should be particularly useful in situations where detection probabilities are heterogeneous among colony sites. The general methodology is illustrated using presence-absence data on two species of herons (Purple Heron, Ardea purpurea and Grey Heron, Ardea cinerea). Estimates of the extinction and colonization rates showed interspecific differences and strong temporal and spatial variations. We were also able to test specific predictions about colony dynamics based on ideas about habitat change and metapopulation dynamics. We recommend estimators based on probabilistic modelling for future work on colony dynamics. We also believe that this methodological framework has wide application to problems in animal

  1. Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

    PubMed Central

    Milgrom, Y M; Murataliev, M B; Boyer, P D

    1998-01-01

    Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid. PMID:9480927

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

    SciTech Connect

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

    2015-06-04

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

  3. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    SciTech Connect

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  4. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

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

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

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

  6. Colony differences in response to trapping in roseate terns

    USGS Publications Warehouse

    Burger, J.; Nisbet, I.C.T.; Zingo, J.M.; Spendelow, J.A.; Safina, C.; Gochfeld, M.

    1995-01-01

    Both members of seabird pairs are normally required to fledge young. Seabirds that nest in sites accessible to predators usually have one parent in attendance during the egg/chick phase. Time devoted to foraging can vary with individual skill and age, prey availability and abundance (Seamy 1978), and distance to foraging grounds (Safina 1990). Although average skill of similar-aged individuals should not vary from colony to colony (Ryder 1980), prey availability and abundance, and spatial distribution of foraging grounds may vary. Thus, the percent of time both members of a pair are present at the nest site may vary in different colonies. In this paper, we examine parental behavior in response to trapping in Roseate Terns (Sterna dougallii) nesting in three of the six major colonies in the northeastern United States: Cedar Beach, New York, Falkner Island, Connecticut; and Bird Island, Massachusetts. Roseate Terns were listed on the United States? Endangered Species List in 1987. We were interested in differences among colonies in how often both parents were present, how soon a mate returned to the nest if one parent was temporarily removed, how soon a trapped bird returned to the nest after release, and the time during which the nest was left unguarded. We feel it is important to recognize and make management decisions based on colony differences where they exist. Our study follows directly from earlier work at Cedar Beach on trapping vulnerability of Common Terns (Sterna hirundo) and Roseate Terns. Nisbet reported that Roseate Terns at Bird Island required about three hours to return to the nest after trapping.

  7. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents

    PubMed Central

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-01-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, aw. The apparent turnover number kcatapp in these systems ranged from 0.2 to 80 s−1 and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing aw. 19F-NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on aw. The rates of NMR-derived fast (k > 107 s−1) and slow (k < 104 s−1) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with kcat. The 19F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant εas, which increased with hydration to εas ≈ 13 in each solvent. In both solvents the transition state free energy data and εas followed Kirkwood’s model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site’s electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  8. Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

    PubMed

    Scaloni, A; Jones, W M; Barra, D; Pospischil, M; Sassa, S; Popowicz, A; Manning, L R; Schneewind, O; Manning, J M

    1992-02-25

    Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions. PMID:1740429

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

    PubMed

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

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed. PMID:25613522

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

    SciTech Connect

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

    2015-03-27

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

  11. Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

    PubMed Central

    Wang, Lu; Fried, Stephen D.; Boxer, Steven G.; Markland, Thomas E.

    2014-01-01

    Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds. PMID:25503367

  12. Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site

    PubMed Central

    Stafford, Kate A.; Palmer III, Arthur G.

    2014-01-01

    Ribonuclease H1 (RNase H) enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold. PMID:25075292

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

    PubMed

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

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst. PMID:27402448

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  16. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    SciTech Connect

    Glantz, C.S.; Ramsdell, J.V.

    1986-04-01

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available.

  17. Activity-dependent labeling of oxygenase enzymes in a trichloroethene-contaminated groundwater site.

    PubMed

    Lee, M Hope; Clingenpeel, Scott C; Leiser, Owen P; Wymore, Ryan A; Sorenson, Kent S; Watwood, Mary E

    2008-05-01

    A variety of naturally occurring bacteria produce enzymes that cometabolically degrade trichloroethene (TCE), including organisms with aerobic oxygenases. Groundwater contaminated with TCE was collected from the aerobic region of the Test Area North site of the Idaho National Laboratory. Samples were evaluated with enzyme activity probes, and resulted in measurable detection of toluene oxygenase activity (6-79% of the total microbial cells). Wells from both inside and outside contaminated plume showed activity. Toluene oxygenase-specific PCR primers determined that toluene-degrading genes were present in all groundwater samples evaluated. In addition, bacterial isolates were obtained and possessed toluene oxygenase enzymes, demonstrated activity, and were dominated by the phylotype Pseudomonas. This study demonstrated, through the use of enzymatic probes and oxygenase gene identification, that indigenous microorganisms at a contaminated site were cometabolically active. Documentation such as this can be used to substantiate observations of natural attenuation of TCE-contaminated groundwater plumes. PMID:17904715

  18. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.

    PubMed

    Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V; Vorobjev, Yuri N; Fedorova, Olga S; Zharkov, Dmitry O

    2013-10-01

    8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation. PMID:23955443

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

    SciTech Connect

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

    1994-06-01

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

  20. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

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

    PubMed

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

    2016-08-18

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(ii), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species-α-Fe(ii) and α-O-are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive 'spectator' iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(ii) to be a mononuclear, high-spin, square planar Fe(ii) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(iv)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function-producing what is known in the context of metalloenzymes as an 'entatic' state-might be a useful way to tune the activity of heterogeneous catalysts. PMID:27535535

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

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

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

  3. Natal origins of migratory monarch butterflies at wintering colonies in Mexico: new isotopic evidence.

    PubMed

    Wassenaar, L I; Hobson, A

    1998-12-22

    Each year, millions of monarch butterflies from eastern North America migrate to overwinter in 10-13 discrete colonies located in the Oyamel forests of central Mexico. For decades efforts to track monarch migration have relied on observations and tag-recapture methods, culminating with the discovery of the wintering colonies in 1975. Monarch tag returns from Mexico, however, are few and primarily from two accessible colonies, and therefore tag-recapture techniques have not quantified natal origins or distinctiveness among monarch populations at wintering sites. Such information would be invaluable in the conservation of the monarch and its migration phenomenon since the wintering sites currently are threatened by habitat alteration. Here we show that stable hydrogen (deltaD) and carbon (delta13C) isotope ratios of wintering monarchs can be used to evaluate natal origins on the summer breeding range. Stable-hydrogen and carbon isotopic values of 597 wintering monarchs from 13 wintering roost sites were compared with isotopic patterns measured in individuals at natal sites across their breeding range over a single migration cycle. We determined that all monarch wintering colonies were composed of individuals originating mainly from the Midwest, United States, thereby providing evidence for a panmictic model of wintering colony composition. However, two colonies showed more northerly origins, suggesting possible priority colonies for conservation efforts. PMID:9860986

  4. Monitoring of geological activity on astronomical sites of the Canary Islands, Hawaii, and Chile

    NASA Astrophysics Data System (ADS)

    Eff-Darwich, Antonio; Garcia-Lorenzo, Begoña; Rodriguez-Losada, Jose A.; Hernández-Gutiérrez, Luis E.; de la Nuez, Julio; Romero-Ruiz, Maria C.

    2009-09-01

    Future large and extremely large ground-based telescopes will demand stable geological settings.Remote sensing could be an unvaluable tool to analyse the impact of geological activity at selected astronomical sites, namely the observatories of El Teide (Tenerife, Canary Islands), Roque de los Muchachos (La Palma, Canary Islands), Mauna Kea (Hawaii) and Paranal (Chile; the candidate site of Cerro Ventarrones, Chile). In this sense, the extent of lava flows, eruptive clouds or ground deformation associated to seismic and/or volcanic activity could be analysed and characterised through remote sensing.

  5. Colony Polymerase Chain Reaction with Schizosaccharomyces pombe.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid "colony PCR" approach may be used. Numerous colony PCR protocols are available, and fundamental to them all is that the colony must be fresh (grown overnight) and that as few cells as possible are used. In this protocol, we present three reliable methods for preparing S. pombe cells for colony PCR. PMID:27140919

  6. Fractal scaling of microbial colonies affects growth

    NASA Astrophysics Data System (ADS)

    Károlyi, György

    2005-03-01

    The growth dynamics of filamentary microbial colonies is investigated. Fractality of the fungal or actinomycetes colonies is shown both theoretically and in numerical experiments to play an important role. The growth observed in real colonies is described by the assumption of time-dependent fractality related to the different ages of various parts of the colony. The theoretical results are compared to a simulation based on branching random walks.

  7. Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity.

    PubMed Central

    Wilson, N J; Moss, S T; Csar, X F; Ward, A C; Hamilton, J A

    1999-01-01

    Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A. PMID:10215588

  8. Monoclonal antibody against macrophage colony-stimulating factor suppresses circulating monocytes and tissue macrophage function but does not alter cell infiltration/activation in cutaneous lesions or clinical outcomes in patients with cutaneous lupus erythematosus.

    PubMed

    Masek-Hammerman, K; Peeva, E; Ahmad, A; Menon, S; Afsharvand, M; Peng Qu, R; Cheng, J B; Syed, J; Zhan, Y; O'Neil, S P; Pleasic-Williams, S; Cox, L A; Beidler, D

    2016-02-01

    This study's objective was to assess the effects of PD-0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony-stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD-0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre- and post-treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14(+) CD16(+) monocytes, urinary N-terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater-than-dose-proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14(+) CD16(+) cells, uNTX, ALT, AST and CK levels at most time-points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between-group differences were seen in CLASI. Patients receiving PD-0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD-0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end-points. PMID:26376111

  9. Breeding ecology of Caspian terns at colonies on the Columbia Plateau

    USGS Publications Warehouse

    Antolos, M.; Roby, D.D.; Collis, K.

    2005-01-01

    We investigated the breeding ecology and diet of Caspian terns on the Columbia Plateau in southeastern Washington and northeastern Oregon. We examined trends in colony size and area during 1996-2001, and estimated number of breeding pairs, nesting density, fledging success, and diet composition at selected colony sites in 2000 and 2001. We found six tern colonies totaling ???1,000 breeding pairs, ranging in size from < 50 to nearly 700 pairs. Predation by mink caused complete abandonment of one of these colonies in 2000 and 2001. The relocation of ???9,000 Caspian tern breeding pairs from Rice Island to East Sand Island in the Columbia River estuary did not result in an obvious increase in the number of tern breeding pairs on the Columbia Plateau during the study period. The majority of Caspian tern prey items at colonies on the mid-Columbia River consisted of juvenile salmonids. At a colony in Potholes Reservoir, Washington, Caspian terns commuted over 100 km round-trip to the Columbia River to forage on juvenile salmonids, suggesting that locally abundant food may be limiting. High nesting densities at other mid-Columbia River colonies suggest that availability of breeding habitat may limit colony size. The small size of Caspian tern colonies on the Columbia Plateau, and possible constraints on availability of suitable nesting habitat within the study area, suggest that the level of predation on ESA-listed juvenile salmonids in this region will likely remain well below that currently observed in the Columbia River estuary.

  10. Defect-Mediated Morphologies in Growing Cell Colonies

    NASA Astrophysics Data System (ADS)

    Doostmohammadi, Amin; Thampi, Sumesh P.; Yeomans, Julia M.

    2016-07-01

    Morphological trends in growing colonies of living cells are at the core of physiological and evolutionary processes. Using active gel equations, which include cell division, we show that shape changes during the growth can be regulated by the dynamics of topological defects in the orientation of cells. The friction between the dividing cells and underlying substrate drives anisotropic colony shapes toward more isotropic morphologies, by mediating the number density and velocity of topological defects. We show that the defects interact with the interface at a specific interaction range, set by the vorticity length scale of flows within the colony, and that the cells predominantly reorient parallel to the interface due to division-induced active stresses.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-01-01

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites. PMID:26853450

  14. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

    NASA Astrophysics Data System (ADS)

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

    2013-05-01

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

  15. Small activating RNA binds to the genomic target site in a seed-region-dependent manner

    PubMed Central

    Meng, Xing; Jiang, Qian; Chang, Nannan; Wang, Xiaoxia; Liu, Chujun; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

    2016-01-01

    RNA activation (RNAa) is the upregulation of gene expression by small activating RNAs (saRNAs). In order to investigate the mechanism by which saRNAs act in RNAa, we used the progesterone receptor (PR) gene as a model, established a panel of effective saRNAs and assessed the involvement of the sense and antisense strands of saRNA in RNAa. All active saRNAs had their antisense strand effectively incorporated into Ago2, whereas such consistency did not occur for the sense strand. Using a distal hotspot for saRNA targeting at 1.6-kb upstream from the PR transcription start site, we further established that gene activation mediated by saRNA depended on the complementarity of the 5′ region of the antisense strand, and that such activity was largely abolished by mutations in this region of the saRNA. We found markedly reduced RNAa effects when we created mutations in the genomic target site of saRNA PR-1611, thus providing evidence that RNAa depends on the integrity of the DNA target. We further demonstrated that this saRNA bound the target site on promoter DNA. These results demonstrated that saRNAs work via an on-site mechanism by binding to target genomic DNA in a seed-region-dependent manner, reminiscent of miRNA-like target recognition. PMID:26873922

  16. A Ty1 Reverse Transcriptase Active-Site Aspartate Mutation Blocks Transposition but Not Polymerization†

    PubMed Central

    Uzun, Ozcan; Gabriel, Abram

    2001-01-01

    Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D211N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme. PMID:11413300

  17. DNA binding induces active site conformational change in the human TREX2 3'-exonuclease.

    PubMed

    de Silva, Udesh; Perrino, Fred W; Hollis, Thomas

    2009-04-01

    The TREX enzymes process DNA as the major 3'-->5' exonuclease activity in mammalian cells. TREX2 and TREX1 are members of the DnaQ family of exonucleases and utilize a two metal ion catalytic mechanism of hydrolysis. The structure of the dimeric TREX2 enzyme in complex with single-stranded DNA has revealed binding properties that are distinct from the TREX1 protein. The TREX2 protein undergoes a conformational change in the active site upon DNA binding including ordering of active site residues and a shift of an active site helix. Surprisingly, even when a single monomer binds DNA, both monomers in the dimer undergo the structural rearrangement. From this we have proposed a model for DNA binding and 3' hydrolysis for the TREX2 dimer. The structure also shows how TREX proteins potentially interact with double-stranded DNA and suggest features that might be involved in strand denaturation to provide a single-stranded substrate for the active site. PMID:19321497

  18. A facile reflux procedure to increase active surface sites form highly active and durable supported palladium@platinum bimetallic nanodendrites

    NASA Astrophysics Data System (ADS)

    Wang, Qin; Li, Yingjun; Liu, Baocang; Xu, Guangran; Zhang, Geng; Zhao, Qi; Zhang, Jun

    2015-11-01

    A series of well-dispersed bimetallic Pd@Pt nanodendrites uniformly supported on XC-72 carbon black are fabricated by using different capping agents. These capping agents are essential for the branched morphology control. However, the surfactant adsorbed on the nanodendrites surface blocks the access of reactant molecules to the active surface sites, and the catalytic activities of these bimetallic nanodendrites are significantly restricted. Herein, a facile reflux procedure to effectively remove the capping agent molecules without significantly affecting their sizes is reported for activating supported nanocatalysts. More significantly, the structure and morphology of the nanodendrites can also be retained, enhancing the numbers of active surface sites, catalytic activity and stability toward methanol and ethanol electro-oxidation reactions. The as-obtained hot water reflux-treated Pd@Pt/C catalyst manifests superior catalytic activity and stability both in terms of surface and mass specific activities, as compared to the untreated catalysts and the commercial Pt/C and Pd/C catalysts. We anticipate that this effective and facile removal method has more general applicability to highly active nanocatalysts prepared with various surfactants, and should lead to improvements in environmental protection and energy production.

  19. Role of methionine in the active site of alpha-galactosidase from Trichoderma reesei.

    PubMed Central

    Kachurin, A M; Golubev, A M; Geisow, M M; Veselkina, O S; Isaeva-Ivanova, L S; Neustroev, K N

    1995-01-01

    alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide. Images Figure 10 PMID:8948456

  20. Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

    PubMed

    Gajadeera, Chathurada S; Zhang, Xinyi; Wei, Yinan; Tsodikov, Oleg V

    2015-02-01

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme. PMID:25576794

  1. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

    PubMed Central

    Hoffmann, Bernd; Mitchell, G. Thomas; Gendron, Patrick; Major, François; Andersen, Angela A.; Collins, Richard A.; Legault, Pascale

    2003-01-01

    Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. PMID:12782785

  2. Immobilized low-activity waste site borehole 299-E17-21

    SciTech Connect

    Reidel, S.P.; Reynolds, K.D.; Horton, D.G.

    1998-08-01

    The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area.

  3. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

    PubMed

    Saam, Jan; Ivanov, Igor; Walther, Matthias; Holzhütter, Hermann-Georg; Kuhn, Hartmut

    2007-08-14

    Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D free-energy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics. PMID:17675410

  4. CO Oxidation on Au/TiO2: Condition-Dependent Active Sites and Mechanistic Pathways.

    PubMed

    Wang, Yang-Gang; Cantu, David C; Lee, Mal-Soon; Li, Jun; Glezakou, Vassiliki-Alexandra; Rousseau, Roger

    2016-08-24

    We present results of ab initio electronic structure and molecular dynamics simulations (AIMD), as well as a microkinetic model of CO oxidation catalyzed by TiO2 supported Au nanocatalysts. A coverage-dependent microkinetic analysis, based on energetics obtained with density functional methods, shows that the dominant kinetic pathway, activated oxygen species, and catalytic active sites are all strongly depended on both temperature and oxygen partial pressure. Under oxidizing conditions and T < 400 K, the prevalent pathway involves a dynamic single atom catalytic mechanism. This reaction is catalyzed by a transient Au-CO species that migrates from the Au-cluster onto a surface oxygen adatom. It subsequently reacts with the TiO2 support via a Mars van Krevelen mechanism to form CO2 and finally the Au atom reintegrates back into the gold cluster to complete the catalytic cycle. At 300 ≤ T ≤ 600 K, oxygen-bound single Oad-Au(+)-CO sites and the perimeter Au-sites of the nanoparticle work in tandem to optimally catalyze the reaction. Above 600 K, a variety of alternate pathways associated with both single-atom and the perimeter sites of the Au nanoparticle are found to be active. Under low oxygen pressures, Oad-Au(+)-CO species can be a source of catalyst deactivation and the dominant pathway involves only Au-perimeter sites. A detailed comparison of the current model and the existing literature resolves many apparent inconsistencies in the mechanistic interpretations. PMID:27480512

  5. Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.

    PubMed

    Tepper, A D; Dammann, H; Bominaar, A A; Véron, M

    1994-12-23

    Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Moréra, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Véron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase. PMID:7798215

  6. Directing reaction pathways by catalyst active-site selection using self-assembled monolayers.

    PubMed

    Pang, Simon H; Schoenbaum, Carolyn A; Schwartz, Daniel K; Medlin, J Will

    2013-01-01

    One key route for controlling reaction selectivity in heterogeneous catalysis is to prepare catalysts that exhibit only specific types of sites required for desired product formation. Here we show that alkanethiolate self-assembled monolayers with varying surface densities can be used to tune selectivity to desired hydrogenation and hydrodeoxygenation products during the reaction of furfural on supported palladium catalysts. Vibrational spectroscopic studies demonstrate that the selectivity improvement is achieved by controlling the availability of specific sites for the hydrogenation of furfural on supported palladium catalysts through the selection of an appropriate alkanethiolate. Increasing self-assembled monolayer density by controlling the steric bulk of the organic tail ligand restricts adsorption on terrace sites and dramatically increases selectivity to desired products furfuryl alcohol and methylfuran. This technique of active-site selection simultaneously serves both to enhance selectivity and provide insight into the reaction mechanism. PMID:24025780

  7. Lessons learned from DOE site culture change activities: Implications for waste management organizations

    SciTech Connect

    Kurstedt, H.A. Jr.; Howard, E.M.; Doss, A.R.; Mallak, L.A.

    1991-01-01

    Management Systems Laboratories (MSL) has worked with the US Department of Energy (DOE) and several of its contractors as they understand and assess the DOE culture change and change the contractor culture to serve DOE's needs. Primarily, these contractors have been those whose responsibilities include starting up and operating weapons materials facilities. The number and scope of these activities have escalated and expanded to contractors at DOE sites such as Westinghouse at the Savannah River Site (SRS) in Aiken, South Carolina, EG G at the Rocky Flats Plant (RFP) in Golden, Colorado, and Westinghouse at the Feed Materials Processing Center (FMPC) in Fernald, Ohio. The point of this paper is not to compare or contrast the relative merit of one site over another. It is to show the lessons, good and bad, and use and communicate those lessons, especially those lessons transferable to other sites in similar situations. 8 refs., 1 fig.

  8. One Kilogram Interstellar Colony Mission

    NASA Astrophysics Data System (ADS)

    Mole, A.

    Small interstellar colony probes based on nanotechnology will become possible long before giant multi-generation ships become affordable. A beam generator and magnetic sail can accelerate a one kg probe to .1 c, braking via the interstellar field can decelerate it, and the field in a distant solar system can allow it to maneuver to an extrasolar planet. A heat shield is used for landing and nanobots emerge to build ever-larger robots and construct colony infrastructure. Humans can then be generated from genomes stored as data in computer memory. Technology is evolving towards these capabilities and should reach the required level in fifty years. The plan appears to be affordable, with the principal cost being the beam generator, estimated at $17 billion.

  9. Nest- and colony-mate recognition in polydomous colonies of meat ants ( Iridomyrmex purpureus)

    NASA Astrophysics Data System (ADS)

    van Wilgenburg, E.; Ryan, D.; Morrison, P.; Marriott, P. J.; Elgar, M. A.

    2006-07-01

    Workers of polydomous colonies of social insects must recognize not only colony-mates residing in the same nest but also those living in other nests. We investigated the impact of a decentralized colony structure on colony- and nestmate recognition in the polydomous Australian meat ant ( Iridomyrmex purpureus). Field experiments showed that ants of colonies with many nests were less aggressive toward alien conspecifics than those of colonies with few nests. In addition, while meat ants were almost never aggressive toward nestmates, they were frequently aggressive when confronted with an individual from a different nest within the same colony. Our chemical analysis of the cuticular hydrocarbons of workers using a novel comprehensive two-dimensional gas chromatography technique that increases the number of quantifiable compounds revealed both colony- and nest-specific patterns. Combined, these data indicate an incomplete transfer of colony odor between the nests of polydomous meat ant colonies.

  10. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  11. Recent Experience Using Active Love Wave Techniques to Characterize Seismographic Station Sites

    NASA Astrophysics Data System (ADS)

    Martin, A. J.; Yong, A.; Salomone, L.

    2014-12-01

    Active-source Love waves recorded by the multi-channel analysis of surface wave (MASLW) technique were recently analyzed in two site characterization projects. Between 2010 and 2011, the 2009 American Recovery and Reinvestment Act (ARRA) funded GEOVision to conduct geophysical investigations at 189 seismographic stations—185 in California and 4 in the Central Eastern U.S. (CEUS). The original project plan was to utilize active and passive Rayleigh wave-based techniques to obtain shear-wave velocity (VS) profiles to a minimum depth of 30 m and the time-averaged VS of the upper 30 meters (VS30). Early in the investigation it became evident that Rayleigh wave techniques, such as multi-channel analysis of surface waves (MASRW), were not effective at characterizing all sites. Shear-wave seismic refraction and MASLW techniques were therefore applied. The MASLW technique was deployed at a total of 38 sites, in addition to other methods, and used as the primary technique to characterize 22 sites, 5 of which were also characterized using Rayleigh wave techniques. In 2012, the Electric Power Research Institute funded characterization of 33 CEUS station sites. Based on experience from the ARRA investigation, both MASRW and MASLW data were acquired by GEOVision at 24 CEUS sites—the remaining 9 sites and 2 overlapping sites were characterized by University of Texas, Austin. Of the 24 sites characterized by GEOVision, 16 were characterized using MASLW data, 4 using both MASLW and MASRW data and 4 using MASRW data. Love wave techniques were often found to perform better, or at least yield phase velocity data that could be more readily modeled using the fundamental mode assumption, at shallow rock sites, sites with steep velocity gradients, and, sites with a thin, low velocity, surficial soil layer overlying stiffer sediments. These types of velocity structure often excite dominant higher modes in Rayleigh wave data, but not in Love wave data. At such sites, it may be possible

  12. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

    PubMed

    Katane, Masumi; Saitoh, Yasuaki; Hanai, Toshihiko; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2010-10-01

    D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO. PMID:20603179

  13. Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

    PubMed

    Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

    2016-07-19

    The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes

  14. Archaeological Activity Report: Post-Review Discoveries Within 45BN431 at Solid Waste Site 128-F-2

    SciTech Connect

    T. E. Marceau; J. J. Sharpe

    2006-12-21

    During monitoring of remedial activities at Solid Waste Site 128-F-2 on August 19, 2005, a concentration of mussel shell was discovered in the west wall of a trench in the northen section of the waste site.

  15. A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1

    PubMed Central

    Tanaka, Takeshi; Fujiwara, Shinsuke; Nishikori, Shingo; Fukui, Toshiaki; Takagi, Masahiro; Imanaka, Tadayuki

    1999-01-01

    We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA. PMID:10583986

  16. Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II).

    PubMed Central

    Nic a'Bháird, N; Yankovskaya, V; Ramsay, R R

    1998-01-01

    The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pKa (7.1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1mol of histidine was modified per mol of CPT-II.When COT was treated with DEPC the kinetics of inhibition were biphasic with an initial rapid loss of activity followed by a slower loss of activity. The residue reacting in the faster phase of inhibition was not a histidine but possibly a serine. The modification of this residue did not lead to complete loss of activity suggesting that a direct role in catalysis is unlikely. It was deduced that the residue modified by DEPC in the slower phase was a lysine and indeed fluorodinitrobenzene (FDNB) inactivated COT with linear pseudo-first-order kinetics. The COT peptide containing the FDNB-labelled lysine was isolated and sequenced. Alignment of this sequence placed it 10 amino acids downstream of the putative active-site histidine. PMID:9480926

  17. Identification of active sites in gold-catalyzed hydrogenation of acrolein.

    PubMed

    Mohr, Christian; Hofmeister, Herbert; Radnik, Jörg; Claus, Peter

    2003-02-19

    The active sites of supported gold catalysts, favoring the adsorption of C=O groups of acrolein and subsequent reaction to allyl alcohol, have been identified as edges of gold nanoparticles. After our recent finding that this reaction preferentially occurs on single crystalline particles rather than multiply twinned ones, this paper reports on a new approach to distinguish different features of the gold particle morphology. Elucidation of the active site issue cannot be simply done by varying the size of gold particles, since the effects of faceting and multiply twinned particles may interfere. Therefore, modification of the gold particle surface by indium has been used to vary the active site characteristics of a suitable catalyst, and a selective decoration of gold particle faces has been observed, leaving edges free. This is in contradiction to theoretical predictions, suggesting a preferred occupation of the low-coordinated edges of the gold particles. On the bimetallic catalyst, the desired allyl alcohol is the main product (selectivity 63%; temperature 593 K, total pressure p(total) = 2 MPa). From the experimentally proven correlation between surface structure and catalytic behavior, the edges of single crystalline gold particles have been identified as active sites for the preferred C=O hydrogenation. PMID:12580618

  18. Strategies and Activities for Using Local Communities as Environmental Education Sites.

    ERIC Educational Resources Information Center

    Roth, Charles E.; Lockwood, Linda G.

    Presented are over 100 environmental education activities which use the local community for a learning site and resource. These lessons are grouped under seven topical headings: (1) biological neighbors, (2) physical environs, (3) built environs, (4) social environs, (5) understanding ourselves, (6) influencing change, and (7) improvement and…

  19. 77 FR 5830 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-06

    ... FR 30,616) of the EA for Issuance of Leases for Wind Resource Data Collection on the Outer... (NOA) in the Federal Register (72 FR 62,672) of the Programmatic EIS for Alternative Energy Development... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the...

  20. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Contracts for Superfund Response Actions Combining Cooperative Agreements § 35.6260 Combining...

  1. Organized Agents: Canadian Teacher Unions as Alternative Sites for Social Justice Activism

    ERIC Educational Resources Information Center

    Rottmann, Cindy

    2008-01-01

    Historically teachers' federations have been some of the major organizational sites for social justice leadership in K-12 public education. Despite this history of activism, social justice teacher unionism remains a relatively underdeveloped concept. This article merges four philosophical conceptions of social justice in education: liberal…

  2. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    PubMed Central

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  3. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 1 2014-07-01 2014-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  4. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  5. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 1 2013-07-01 2013-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  6. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 1 2012-07-01 2012-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  7. The Thumbs Up Ecology Curriculum: A Fun Group of School Site Activities for Sixth Graders.

    ERIC Educational Resources Information Center

    Smith, John; And Others

    This guide is a collection of "fun" school site activities for sixth graders. Some of the topics covered are: animals, trees, energy and lifestyle, land use and you, energy conservation, and car-pooling. Each section offers both introductory information about the topic as well as questions to ponder such as what, so what, now what, and another way…

  8. IN VIVO ACTIVITY OF RHOPALOSIPHUM PADI VIRUS INTERNAL RIBOSOME ENTRY SITES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The RNA genome of Rhopalosiphum padi virus (RhPV), like other members of the Dicistroviridae, contains two open reading frames that are preceded by internal ribosome entry sites (IRESs). To compare the activities of the two RhPV IRESs in insect cells, a system was established for the in vivo transc...

  9. Cyclic silicate active site and stereochemical match for apatite nucleation on pseudowollastonite bioceramic-bone interfaces.

    PubMed

    Sahai, Nita; Anseau, Michel

    2005-10-01

    Hydroxyapatite (Ca5(PO4)3(OH)) forms on pseudowollastonite (psW) (alpha-CaSiO3) in vitro in simulated body fluid, human parotid saliva and cell-culture medium, and in vivo in implanted rat tibias. We used crystallographic constraints with ab initio molecular orbital calculations to identify the active site and reaction mechanism for heterogeneous nucleation of the earliest calcium phosphate oligomer/phase. The active site is the planar, cyclic, silicate trimer (Si3O9) on the (001) face of psW. The trimer has three silanol groups (>SiOH) arranged at 60 degrees from each other, providing a stereochemical match for O atoms bonded to Ca2+ on the (001) face of hydroxyapatite. Calcium phosphate nucleation is modeled in steps as hydrolysis of surface Ca-O bonds with leaching of Ca2+ into solution, protonation of the surface Si-O groups to form silanols, calcium sorption as an inner-sphere surface complex and, attachment of HPO4(2-). Our model explains the experimental solution and high resolution transmission electron microscopy data for epitaxial hydroxyapatite growth on psW in vitro and in vivo. We propose that the cyclic silicate trimer is the universal active site for heterogeneous, stereochemically promoted nucleation on silicate-based bioactive ceramics. A critical active site-density and a point of zero charge of the bioceramic less than physiological pH are required for bioactivity. PMID:15949543

  10. Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

    PubMed

    Kleinboelting, Silke; Ramos-Espiritu, Lavoisier; Buck, Hannes; Colis, Laureen; van den Heuvel, Joop; Glickman, J Fraser; Levin, Lonny R; Buck, Jochen; Steegborn, Clemens

    2016-04-29

    The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs. PMID:26961873

  11. Intercellular Genomics of Subsurface Microbial Colonies

    SciTech Connect

    Ortoleva, Peter; Tuncay, Kagan; Gannon, Dennis; Meile, Christof

    2007-02-14

    This report summarizes progress in the second year of this project. The objective is to develop methods and software to predict the spatial configuration, properties and temporal evolution of microbial colonies in the subsurface. To accomplish this, we integrate models of intracellular processes, cell-host medium exchange and reaction-transport dynamics on the colony scale. At the conclusion of the project, we aim to have the foundations of a predictive mathematical model and software that captures the three scales of these systems – the intracellular, pore, and colony wide spatial scales. In the second year of the project, we refined our transcriptional regulatory network discovery (TRND) approach that utilizes gene expression data along with phylogenic similarity and gene ontology analyses and applied it successfully to E.coli, human B cells, and Geobacter sulfurreducens. We have developed a new Web interface, GeoGen, which is tailored to the reconstruction of microbial TRNs and solely focuses on Geobacter as one of DOE’s high priority microbes. Our developments are designed such that the frameworks for the TRND and GeoGen can readily be used for other microbes of interest to the DOE. In the context of modeling a single bacterium, we are actively pursuing both steady-state and kinetic approaches. The steady-state approach is based on a flux balance that uses maximizing biomass growth rate as its objective, subjected to various biochemical constraints, for the optimal values of reaction rates and uptake/release of metabolites. For the kinetic approach, we use Karyote, a rigorous cell model developed by us for an earlier DOE grant and the DARPA BioSPICE Project. We are also investigating the interplay between bacterial colonies and environment at both pore and macroscopic scales. The pore scale models use detailed representations for realistic porous media accounting for the distribution of grain size whereas the macroscopic models employ the Darcy-type flow

  12. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

    PubMed

    Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

    2013-05-10

    The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. PMID:23564727

  13. Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

    PubMed Central

    Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

    2013-01-01

    Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

  14. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  15. Functional copper at the acetyl-CoA synthase active site

    PubMed Central

    Seravalli, Javier; Gu, Weiwei; Tam, Annie; Strauss, Erick; Begley, Tadhg P.; Cramer, Stephen P.; Ragsdale, Stephen W.

    2003-01-01

    The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood–Ljungdahl pathway of autotrophic CO2 fixation. A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M. thermoacetica ACS active site is essential. Depletion of copper correlates with reduction in ACS activity and in intensity of the “NiFeC” EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH. In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity. Cu is also found in the methanogenic CODH/ACS from Methanosarcina thermophila. XAS studies are consistent with a distorted Cu(I)–S3 site in the fully active enzyme in solution. Cu extended x-ray absorption fine structure analysis indicates an average Cu–S bond length of 2.25 Å and a metal neighbor at 2.65 Å, consistent with the Cu–Ni distance observed in the crystal structure. XAS experiments in the presence of seleno-CoA reveal a Cu–S3Se environment with a 2.4-Å Se–Cu bond, strongly implicating a Cu–SCoA intermediate in the mechanism of acetyl-CoA synthesis. These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms. PMID:12589021

  16. Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase.

    PubMed

    Chen, Mengbin; Chou, Wayne K W; Al-Lami, Naeemah; Faraldos, Juan A; Allemann, Rudolf K; Cane, David E; Christianson, David W

    2016-05-24

    Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule coordinates to the Mg(2+)C ion. There is no obvious role for water in the ATAS mechanism because the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg(2+)C-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity of Mg(2+)C-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates. PMID:27172425

  17. Deadly competition between sibling bacterial colonies

    NASA Astrophysics Data System (ADS)

    Be'Er, Avraham

    2011-03-01

    As a result of stress due to nutrient limitation or antibiotics, competing individual bacteria within a single colony may lyse sibling cells to release nutrients (cannibalism) or DNA (fratricide). However, we have recently shown that competition is not limited to individuals, but can occur at the colony level [A. Be'er et al., PNAS 106, 428 (2009); A. Be'er et al., PNAS 107, 6258 (2010).] In response to the presence of an encroaching sibling colony, Paenibacillus dendritiformis bacteria secrete a lethal protein, lysing cells at the interface between the colonies. Analysis of the proteins secreted by these competing sibling colonies, combined with a mathematical model, shows how colonies maintain their growth by self-regulating the secretion of two proteins: subtilisin (a well-known growth promoter), and Slf (a previously unknown protein, which is lethal). The results also explain why a single colony is not inhibited by its own secretions.

  18. Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O2 Activation

    PubMed Central

    2015-01-01

    Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes. PMID

  19. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  20. Keratin subsidies promote feather decomposition via an increase in keratin-consuming arthropods and microorganisms in bird breeding colonies

    NASA Astrophysics Data System (ADS)

    Sugiura, Shinji; Masuya, Hayato

    2015-06-01

    Resource subsidies are well known to increase population densities of consumers. The decomposition process of these subsidised resources can be influenced by increasing consumer abundance. However, few studies have assessed whether resource subsidies can promote resource decomposition via a population increase in consumers. Here, we examined the effects of keratin subsidies on feather decomposition in egret and heron breeding colonies. Egrets and herons (Ardeidae) frequently breed in inland forests and provide large amounts of keratin materials to the forest floor in the form of feathers of chicks (that die). We compared the decrease in the weights of egret and heron feathers (experimentally placed on the forest floor) over a 12-month period among egret/heron breeding colonies (five sites) and areas outside of colonies (five sites) in central Japan. Of the feathers placed experimentally on forest floors, 92-97 % and 99-100 % in colonies and 47-50 % and 71-90 % in non-colony areas were decomposed after 4 and 12 months, respectively. Then, decomposition rates of feathers were faster in colonies than in areas outside of colonies, suggesting that keratin subsidies can promote feather decomposition in colonies. Field observations and laboratory experiments indicated that keratin-feeding arthropods and keratinophilic fungi played important roles in feather decomposition. Therefore, scavenging arthropods and keratinophilic fungi, which dramatically increased in egret and heron breeding colonies, could accelerate the decomposition of feathers supplied to the forest floor of colonies.

  1. Keratin subsidies promote feather decomposition via an increase in keratin-consuming arthropods and microorganisms in bird breeding colonies.

    PubMed

    Sugiura, Shinji; Masuya, Hayato

    2015-06-01

    Resource subsidies are well known to increase population densities of consumers. The decomposition process of these subsidised resources can be influenced by increasing consumer abundance. However, few studies have assessed whether resource subsidies can promote resource decomposition via a population increase in consumers. Here, we examined the effects of keratin subsidies on feather decomposition in egret and heron breeding colonies. Egrets and herons (Ardeidae) frequently breed in inland forests and provide large amounts of keratin materials to the forest floor in the form of feathers of chicks (that die). We compared the decrease in the weights of egret and heron feathers (experimentally placed on the forest floor) over a 12-month period among egret/heron breeding colonies (five sites) and areas outside of colonies (five sites) in central Japan. Of the feathers placed experimentally on forest floors, 92-97 % and 99-100 % in colonies and 47-50 % and 71-90 % in non-colony areas were decomposed after 4 and 12 months, respectively. Then, decomposition rates of feathers were faster in colonies than in areas outside of colonies, suggesting that keratin subsidies can promote feather decomposition in colonies. Field observations and laboratory experiments indicated that keratin-feeding arthropods and keratinophilic fungi played important roles in feather decomposition. Therefore, scavenging arthropods and keratinophilic fungi, which dramatically increased in egret and heron breeding colonies, could accelerate the decomposition of feathers supplied to the forest floor of colonies. PMID:25903424

  2. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    PubMed

    Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

    2007-09-01

    Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent. PMID:17673485

  3. Locomotor activity influences muscle architecture and bone growth but not muscle attachment site morphology

    PubMed Central

    Rabey, Karyne N.; Green, David J.; Taylor, Andrea B.; Begun, David R.; Richmond, Brian G.; McFarlin, Shannon C.

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual’s past behaviour. PMID:25467113

  4. Translucent Colony Form of the Gram-Negative, Levan-Producing Bacterium, Aerobacter levanicum

    PubMed Central

    Takeshita, Masaru

    1973-01-01

    A spontaneously occurring variation of Aerobacter levanicum, affecting the appearance of colonies on sucrose agar (opaque to translucent colony form), has been observed and studied. This appears to be a mutation that is accompanied by a significant increase in levansucrase (EC 2.4.1.10) activity, in levan production, and by a change in some of the properties of levansucrase. PMID:4745425

  5. Detection of fungi colony growth on bones by dynamic speckle

    NASA Astrophysics Data System (ADS)

    Vincitorio, F. M.; Budini, N.; Mulone, C.; Spector, M.; Freyre, C.; López Díaz, A. J.; Ramil, A.

    2013-11-01

    In this work we have studied the dynamic speckle patterns of mucor fungi colonies, which were inoculated on different samples. We were interested in analyzing the development of fungi colonies in bones, since during the last two years, a series of infections by mucor fungi have been reported on patients from different hospitals in Argentina. Coincidentally, all of these infections appeared on patients that were subjected to a surgical intervention for implantation of a titanium prosthesis. Apparently, the reason of the infection was a deficient sterilization process in conjunction with an accidental contamination. We observed that fungi growth, activity and death can be distinguished by means of the dynamic speckle technique.

  6. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

    PubMed

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  7. Active site of the replication protein of the rolling circle plasmid pC194.

    PubMed Central

    Noirot-Gros, M F; Bidnenko, V; Ehrlich, S D

    1994-01-01

    Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step. Images PMID:7925284

  8. Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

    PubMed

    Gross, M; Whetzel, N K; Folk, J E

    1975-10-10

    Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate. PMID:240837

  9. Modelled drift patterns of fish larvae link coastal morphology to seabird colony distribution.

    PubMed

    Sandvik, Hanno; Barrett, Robert T; Erikstad, Kjell Einar; Myksvoll, Mari S; Vikebø, Frode; Yoccoz, Nigel G; Anker-Nilssen, Tycho; Lorentsen, Svein-Håkon; Reiertsen, Tone K; Skarðhamar, Jofrid; Skern-Mauritzen, Mette; Systad, Geir Helge

    2016-01-01

    Colonial breeding is an evolutionary puzzle, as the benefits of breeding in high densities are still not fully explained. Although the dynamics of existing colonies are increasingly understood, few studies have addressed the initial formation of colonies, and empirical tests are rare. Using a high-resolution larval drift model, we here document that the distribution of seabird colonies along the Norwegian coast can be explained by variations in the availability and predictability of fish larvae. The modelled variability in concentration of fish larvae is, in turn, predicted by the topography of the continental shelf and coastline. The advection of fish larvae along the coast translates small-scale topographic characteristics into a macroecological pattern, viz. the spatial distribution of top-predator breeding sites. Our findings provide empirical corroboration of the hypothesis that seabird colonies are founded in locations that minimize travel distances between breeding and foraging locations, thereby enabling optimal foraging by central-place foragers. PMID:27173005

  10. Modelled drift patterns of fish larvae link coastal morphology to seabird colony distribution

    NASA Astrophysics Data System (ADS)

    Sandvik, Hanno; Barrett, Robert T.; Erikstad, Kjell Einar; Myksvoll, Mari S.; Vikebø, Frode; Yoccoz, Nigel G.; Anker-Nilssen, Tycho; Lorentsen, Svein-Håkon; Reiertsen, Tone K.; Skarðhamar, Jofrid; Skern-Mauritzen, Mette; Systad, Geir Helge

    2016-05-01

    Colonial breeding is an evolutionary puzzle, as the benefits of breeding in high densities are still not fully explained. Although the dynamics of existing colonies are increasingly understood, few studies have addressed the initial formation of colonies, and empirical tests are rare. Using a high-resolution larval drift model, we here document that the distribution of seabird colonies along the Norwegian coast can be explained by variations in the availability and predictability of fish larvae. The modelled variability in concentration of fish larvae is, in turn, predicted by the topography of the continental shelf and coastline. The advection of fish larvae along the coast translates small-scale topographic characteristics into a macroecological pattern, viz. the spatial distribution of top-predator breeding sites. Our findings provide empirical corroboration of the hypothesis that seabird colonies are founded in locations that minimize travel distances between breeding and foraging locations, thereby enabling optimal foraging by central-place foragers.

  11. Modelled drift patterns of fish larvae link coastal morphology to seabird colony distribution

    PubMed Central

    Sandvik, Hanno; Barrett, Robert T.; Erikstad, Kjell Einar; Myksvoll, Mari S.; Vikebø, Frode; Yoccoz, Nigel G.; Anker-Nilssen, Tycho; Lorentsen, Svein-Håkon; Reiertsen, Tone K.; Skarðhamar, Jofrid; Skern-Mauritzen, Mette; Systad, Geir Helge

    2016-01-01

    Colonial breeding is an evolutionary puzzle, as the benefits of breeding in high densities are still not fully explained. Although the dynamics of existing colonies are increasingly understood, few studies have addressed the initial formation of colonies, and empirical tests are rare. Using a high-resolution larval drift model, we here document that the distribution of seabird colonies along the Norwegian coast can be explained by variations in the availability and predictability of fish larvae. The modelled variability in concentration of fish larvae is, in turn, predicted by the topography of the continental shelf and coastline. The advection of fish larvae along the coast translates small-scale topographic characteristics into a macroecological pattern, viz. the spatial distribution of top-predator breeding sites. Our findings provide empirical corroboration of the hypothesis that seabird colonies are founded in locations that minimize travel distances between breeding and foraging locations, thereby enabling optimal foraging by central-place foragers. PMID:27173005

  12. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  13. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  14. Reliving Colonial Days in Your Classroom. Curriculum Boosters. Social Studies.

    ERIC Educational Resources Information Center

    Hennessey, Gail Skroback

    1994-01-01

    The article presents hands-on classroom projects to teach elementary students about colonial American history. Students make their own natural dyes, cook blueberry slump, and play cup-and-ball the way the colonists did. The activities integrate science, math, history, art, and language arts. (SM)

  15. Isolation of a murine osteoclast colony-stimulating factor.

    PubMed Central

    Lee, M Y; Eyre, D R; Osborne, W R

    1991-01-01

    Cultures of a cell line derived from a murine mammary carcinoma that induces hypercalcemia were examined for soluble products that could induce osteoclasts to differentiate from murine bone marrow cells. The serum-free culture supernatant of this cell line stimulated growth of colonies from bone marrow cells that exhibited tartrate-resistant acid phosphatase (TRAPase) activity. These TRAPase-positive cells demonstrated essential features of osteoclasts when cultured with mineralized bone or dentin. The culture period required for colony development and the frequency of colony-forming cells indicated that relatively primitive marrow progenitors were stimulated by a tumor-derived factor(s) to form immature osteoclasts. Other colony-stimulating factors (CSFs), including granulocyte CSF, macrophage CSF, granulocyte-macrophage CSF and interleukin 3, were ruled out as the source of the activity produced by the tumor cells. The biological activity was successfully purified by gel filtration chromatography and reverse-phase HPLC. By SDS/PAGE, the activity was traced to a protein of approximately 17 kDa. Functional and biochemical studies of the purified factor suggest that it is distinct from any known CSF of myeloid cells. This protein appears to be a CSF for the osteoclast lineage, osteoclast CSF (O-CSF). Images PMID:1924309

  16. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ligands'' with localized surface orbitals perturbed only by these ligands''. These complexes'' are based on a twelve coordinate species with the ligands'' attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  17. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ``ligands`` with localized surface orbitals perturbed only by these ``ligands``. These ``complexes`` are based on a twelve coordinate species with the ``ligands`` attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  18. Evidence for oxygen binding at the active site of particulate methane monooxygenase.

    PubMed

    Culpepper, Megen A; Cutsail, George E; Hoffman, Brian M; Rosenzweig, Amy C

    2012-05-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an α(3)β(3)γ(3) trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit (denoted as spmoB) indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O(2) or H(2)O(2) or pMMO with H(2)O(2), an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the μ-η(2):η(2)-peroxo-Cu(II)(2) species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O(2) binding to the proposed active site. Reaction of the 345 nm species with CH(4) results in the disappearance of the spectroscopic feature, suggesting that this O(2) intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site. PMID:22540911

  19. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

    PubMed

    El Sayyed, Hafez; Le Chat, Ludovic; Lebailly, Elise; Vickridge, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco; Espéli, Olivier

    2016-05-01

    Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle. PMID:27171414

  20. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

    PubMed Central

    Lebailly, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco

    2016-01-01

    Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle. PMID:27171414

  1. Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.

    PubMed

    Lansdell, Stuart J; Sathyaprakash, Chaitra; Doward, Anne; Millar, Neil S

    2015-01-01

    In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies. PMID:25338672

  2. Final Report - Independent Verification Survey Activities at the Seperations Process Research Unit Sites, Niskayuna, New York

    SciTech Connect

    Evan Harpenau

    2011-03-15

    The Separations Process Research Unit (SPRU) complex located on the Knolls Atomic Power Laboratory (KAPL) site in Niskayuna, New York, was constructed in the late 1940s to research the chemical separation of plutonium and uranium (Figure A-1). SPRU operated as a laboratory scale research facility between February 1950 and October 1953. The research activities ceased following the successful development of the reduction oxidation and plutonium/uranium extraction processes. The oxidation and extraction processes were subsequently developed for large scale use by the Hanford and Savannah River sites (aRc 2008a). Decommissioning of the SPRU facilities began in October 1953 and continued through the 1990s.

  3. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    SciTech Connect

    Petersen, C.A., Westinghouse Hanford

    1996-07-17

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level wastes, for disposal in a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  4. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    SciTech Connect

    Petersen, C.A.

    1996-09-20

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level waste, for disposal is a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  5. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  6. Kinetic model of ethopropazine interaction with horse serum butyrylcholinesterase and its docking into the active site.

    PubMed

    Golicnik, Marko; Sinko, Goran; Simeon-Rudolf, Vera; Grubic, Zoran; Stojan, Jure

    2002-02-01

    The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor. PMID:11811945

  7. Controlling activation site density by low-energy far-field stimulation in cardiac tissue

    NASA Astrophysics Data System (ADS)

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites (“virtual electrodes”) in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  8. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

  9. Localization of the active site of an enzyme, bacterial luciferase, using two-quantum affinity modification

    NASA Astrophysics Data System (ADS)

    Benimetskaya, L. Z.; Gitelzon, I. I.; Kozionov, Andrew L.; Novozhilov, S. Y.; Petushkov, V. N.; Rodionova, N. S.; Stockman, Mark I.

    1991-11-01

    For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the (alpha) -subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ((lambda) 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial

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

    SciTech Connect

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

    2013-07-01

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

  11. Differential behavior of the sub-sites of cytochrome 450 active site in binding of substrates, and products (implications for coupling/uncoupling).

    PubMed

    Narasimhulu, Shakunthala

    2007-03-01

    The cytochrome P450 catalyzes hydroxylation of many substrates in the presence of O(2) and specific electron transport system. The ternary complex S-Fe(+)O(2) with substrate and O(2) bound to their respective sites on the reduced enzyme is an important intermediate in the formation of the hydroxylating species. Then the active site may be considered as having two sub-sites geared for entirely different types of functionally relevant interactions. The two sites are the substrate binding site, the specific protein residues (Site I), and the L(6) position of the iron (Site II) to which O(2) binds upon reduction. In the ferric enzyme, when substrate binds to Site I, the low spin six-coordinated P450 is converted to the readily reducible high spin five coordinated state. Certain amines and OH compounds, such as products of P450-catalyzed reactions, can bind to Site II resulting in six coordinated inhibited complexes. Then the substrate and product interactions with the two sub-sites can regulate the functional state of the enzyme during catalysis. Product interactions have received very little attention. CYP101 is the only P450 in which X-ray and spectroscopic data on all three structures, the substrate-free, camphor-bound and the 5-exo-OHcamphor-bound are available. The substrate-free CYP101 is low spin and six-coordinated with a water molecule ligated at the L(6) position of the iron. The substrate camphor binds to Site I, and releases the L(6) water despite its inability to bind to this site, indicating that Site I binding can inhibit Site II ligation. The product 5-exo-OHcamphor in addition to binding to Site I, binds to Site II through its -OH group forming Fe-O bond, resulting in the low spin six-coordinated complex. New temperature-jump relaxation kinetic data indicating that Site II ligation inhibits Site I binding are presented. It appears that the Site I and Site II function as interacting sub-sites. The inhibitory allosteric interactions between the two sub-sites

  12. Spatial Heterogeneity as a Genetic Mixing Mechanism in Highly Philopatric Colonial Seabirds

    PubMed Central

    Cristofari, Robin; Trucchi, Emiliano; Whittington, Jason D.; Vigetta, Stéphanie; Gachot-Neveu, Hélène; Stenseth, Nils Christian; Le Maho, Yvon; Le Bohec, Céline

    2015-01-01

    How genetic diversity is maintained in philopatric colonial systems remains unclear, and understanding the dynamic balance of philopatry and dispersal at all spatial scales is essential to the study of the evolution of coloniality. In the King penguin, Aptenodytes patagonicus, return rates of post-fledging chicks to their natal sub-colony are remarkably high. Empirical studies have shown that adults return year after year to their previous breeding territories within a radius of a few meters. Yet, little reliable data are available on intra- and inter-colonial dispersal in this species. Here, we present the first fine-scale study of the genetic structure in a king penguin colony in the Crozet Archipelago. Samples were collected from individual chicks and analysed at 8 microsatellite loci. Precise geolocation data of hatching sites and selective pressures associated with habitat features were recorded for all sampling locations. We found that despite strong natal and breeding site fidelity, king penguins retain a high degree of panmixia and genetic diversity. Yet, genetic structure appears markedly heterogeneous across the colony, with higher-than-expected inbreeding levels, and local inbreeding and relatedness hotspots that overlap predicted higher-quality nesting locations. This points towards heterogeneous population structure at the sub-colony level, in which fine-scale environmental features drive local philopatric behaviour, while lower-quality patches may act as genetic mixing mechanisms at the colony level. These findings show how a lack of global genetic structuring can emerge from small-scale heterogeneity in ecological parameters, as opposed to the classical model of homogeneous dispersal. Our results also emphasize the importance of sampling design for estimation of population parameters in colonial seabirds, as at high spatial resolution, basic genetic features are shown to be location-dependent. Finally, this study stresses the importance of

  13. Pathogenesis of varroosis at the level of the honey bee (Apis mellifera) colony.

    PubMed

    Wegener, J; Ruhnke, H; Scheller, K; Mispagel, S; Knollmann, U; Kamp, G; Bienefeld, K

    2016-01-01

    The parasitic mite Varroa destructor, in interaction with different viruses, is the main cause of honey bee colony mortality in most parts of the world. Here we studied how effects of individual-level parasitization are reflected by the bee colony as a whole. We measured disease progression in an apiary of 24 hives with differing degree of mite infestation, and investigated its relationship to 28 biometrical, physiological and biochemical indicators. In early summer, when the most heavily infested colonies already showed reduced growth, an elevated ratio of brood to bees, as well as a strong presence of phenoloxidase/prophenoloxidase in hive bees were found to be predictors of the time of colony collapse. One month later, the learning performance of worker bees as well as the activity of glucose oxidase measured from head extracts were significantly linked to the timing of colony collapse. Colonies at the brink of collapse were characterized by reduced weight of winter bees and a strong increase in their relative body water content. Our data confirm the importance of the immune system, known from studies of individually-infested bees, for the pathogenesis of varroosis at colony level. However, they also show that single-bee effects cannot always be extrapolated to the colony as a whole. This fact, together with the prominent role of colony-level factors like the ratio between brood and bees for disease progression, stress the importance of the superorganismal dimension of Varroa research. PMID:27296894

  14. Estimated areal extent of colonies of black-tailed prairie dogs in the northern Great Plains

    USGS Publications Warehouse

    Sidle, John G.; Johnson, D.H.; Euliss, B.R.

    2001-01-01

    During 1997-1998, we undertook an aerial survey, with an aerial line-intercept technique, to estimate the extent of colonies of black-tailed prairie dogs (Cynomys ludovicianus) in the northern Great Plains states of Nebraska, North Dakota, South Dakota, and Wyoming. We stratified the survey based on knowledge of colony locations, computed 2 types of estimates for each stratum, and combined ratio estimates for high-density strata with average density estimates for low-density strata. Estimates of colony areas for black-tailed prairie dogs were derived from the average percentages of lines intercepting prairie dog colonies and ratio estimators. We selected the best estimator based on the correlation between length of transect line and length of intercepted colonies. Active colonies of black-tailed prairie dogs occupied 2,377.8 km2 i?? 186.4 SE, whereas inactive colonies occupied 560.4 i?? 89.2 km2. These data represent the 1st quantitative assessment of prairie-dog colonies in the northern Great Plains. The survey dispels popular notions that millions of square kilometers of colonies of black-tailed prairie dogs exist in the northern Great Plains and can form the basis for future survey efforts

  15. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  16. Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

    PubMed Central

    Hobbs, G Aaron; Gunawardena, Harsha P; Baker, Rachael; Campbell, Sharon L

    2013-01-01

    KRas has recently been shown to be activated by monoubiquitination (mUb). Similar to oncogenic mutations, mUb of Ras at position 147 activates Ras by causing a defect in GTPase activating protein (GAP) function. To characterize the mechanism by which mUb impairs GAP-mediated downregulation of Ras, we made various modifications at position 147 of Ras and examined the impact on Ras sensitivity to GAP function. Whereas small modifications (iodoacetamide and glutathione) at position 147 of Ras do not affect GAP-mediated hydrolysis, ligation of Ras to UbG76C (native linker), UbX77C (one residue longer), and PDZ2 (with a native ubiquitin linker) was defective in GAP-mediated GTP hydrolysis. However, restoration of GAP activity was observed for Ras modified with the PDZ2 domain containing a shorter and stiffer linker region than ubiquitin. Therefore, the properties of the linker region dictate whether modification affects GAP-mediated hydrolysis, and our data indicate that the GAP defect requires a minimum linker length of 7 to 8 residues. PMID:24030601

  17. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  18. Exploration versus exploitation in polydomous ant colonies.

    PubMed

    Cook, Zoe; Franks, Daniel W; Robinson, Elva J H

    2013-04-21

    In socially foraging species resource information can be shared between individuals, increasing foraging success. In ant colonies, nestmate recruitment allows high exploitation rates at known resources however, to maximise foraging efficiency this must be balanced with searching for new resources. Many ant species form colonies inhabiting two or more spatially separated but socially connected nests: this type of organisation is known as polydomy. Polydomous colonies may benefit from increased foraging efficiency by carrying out dispersed-central place foraging. However, decentralisation of the colony may affect recruitment success by limiting interaction between ants based in separate nests. We use an agent-based model which compares the foraging success of monodomous and polydomous colonies in different food environments, incorporating recruitment through pheromone trails and group foraging. In contrast to previous results we show that polydomy is beneficial in some but not all cases. Polydomous colonies discover resources at a higher rate, making them more successful when food is highly dispersed, but their relative success can be lowered by limitations on recruitment success. Monodomous colonies can have higher foraging efficiency than polydomous colonies by exploiting food more rapidly. The results show the importance of interactions between recruitment strategy, colony size, and colony organisation. PMID:23380232

  19. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    SciTech Connect

    1998-07-01

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC`s production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities.

  20. The Histamine N-Methyltransferase T105I Polymorphism Affects Active Site Structure and Dynamics†

    PubMed Central

    Rutherford, Karen; Parson, William W.; Daggett, Valerie

    2010-01-01

    Histamine N-methyltransferase (HNMT) is the sole enzyme responsible for inactivating histamine in the mammalian brain. The human HNMT gene contains a common threonine-isoleucine polymorphism at residue 105, distal from the active site. The 105I variant has decreased activity and lower protein levels relative to the 105T protein. Crystal structures of both variants have been solved, but reveal little regarding how the T105I polymorphism affects activity. We performed molecular dynamics simulations of both 105T and 105I at 37°C to explore the structural and dynamic consequences of the polymorphism. The simulations indicate that replacing Thr with the larger Ile residue leads to greater burial of residue 105 and heightened packing interactions between residue105 and residues within helix α3 and strand β3. This altered packing is directly translated to the active site resulting in the reorientation of several co-substrate-binding residues. The simulations also show that the hydrophobic histamine-binding domain in both proteins undergoes a large-scale breathing motion that exposes key catalytic residues and lessens the hydrophobicity of the substrate-binding site. PMID:18154359

  1. Substrate conformational transitions in the active site of chorismate mutase: Their role in the catalytic mechanism

    PubMed Central

    Guo, Hong; Cui, Qiang; Lipscomb, William N.; Karplus, Martin

    2001-01-01

    Chorismate mutase acts at the first branch-point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. The results of molecular dynamics simulations of the substrate in solution and in the active site of chorismate mutase are reported. Two nonreactive conformers of chorismate are found to be more stable than the reactive pseudodiaxial chair conformer in solution. It is shown by QM/MM molecular dynamics simulations, which take into account the motions of the enzyme, that when these inactive conformers are bound to the active site, they are rapidly converted to the reactive chair conformer. This result suggests that one contribution of the enzyme is to bind the more prevalent nonreactive conformers and transform them into the active form in a step before the chemical reaction. The motion of the reactive chair conformer in the active site calculated by using the QM/MM potential generates transient structures that are closer to the transition state than is the stable CHAIR conformer. PMID:11481470

  2. From single crystal surfaces to single atoms: investigating active sites in electrocatalysis.

    PubMed

    O'Mullane, Anthony P

    2014-04-21

    Electrocatalytic processes will undoubtedly be at the heart of energising future transportation and technology with the added importance of being able to create the necessary fuels required to do so in an environmentally friendly and cost effective manner. For this to be successful two almost mutually exclusive surface properties need to be reconciled, namely producing highly active/reactive surface sites that exhibit long term stability. This article reviews the various approaches which have been undertaken to study the elusive nature of these active sites on metal surfaces which are considered as adatoms or clusters of adatoms with low coordination number. This includes the pioneering studies at extended well defined stepped single crystal surfaces using cyclic voltammetry up to the highly sophisticated in situ electrochemical imaging techniques used to study chemically synthesised nanomaterials. By combining the information attained from single crystal surfaces, individual nanoparticles of defined size and shape, density functional theory calculations and new concepts such as mesoporous multimetallic thin films and single atom electrocatalysts new insights into the design and fabrication of materials with highly active but stable active sites can be achieved. The area of electrocatalysis is therefore not only a fascinating and exciting field in terms of realistic technological and economical benefits but also from the fundamental understanding that can be acquired by studying such an array of interesting materials. PMID:24599277

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

    PubMed

    Foo, Guo Shiou; Sievers, Carsten

    2015-02-01

    The chemical oxidation of activated carbon by H2 O2 and H2 SO4 is investigated, structural and chemical modifications are characterized, and the materials are used as catalysts for the hydrolysis of cellulose. Treatment with H2 O2 enlarges the pore size and imparts functional groups such as phenols, lactones, and carboxylic acids. H2 SO4 treatment targets the edges of carbon sheets primarily, and this effect is more pronounced with a higher temperature. Adsorption isotherms demonstrate that the adsorption of oligomers on functionalized carbon is dominated by van der Waals forces. The materials treated chemically are active for the hydrolysis of cellulose despite the relative weakness of most of their acid sites. It is proposed that a synergistic effect between defect sites and functional groups enhances the activity by inducing a conformational change in the glucan chains if they are adsorbed at defect sites. This activates the glycosidic bonds for hydrolysis by in-plane functional groups. PMID:25504913

  4. Site-dependent catalytic activity of graphene oxides towards oxidative dehydrogenation of propane.

    PubMed

    Tang, Shaobin; Cao, Zexing

    2012-12-28

    Graphene oxides (GOs) may offer extraordinary potential in the design of novel catalytic systems due to the presence of various oxygen functional groups and their unique electronic and structural properties. Using first-principles calculations, we explore the plausible mechanisms for the oxidative dehydrogenation (ODH) of propane to propene by GOs and the diffusion of the surface oxygen-containing groups under an external electric field. The present results show that GOs with modified oxygen-containing groups may afford high catalytic activity for the ODH of propane to propene. The presence of hydroxyl groups around the active sites provided by epoxides can remarkably enhance the C-H bond activation of propane and the activity enhancement exhibits strong site dependence. The sites of oxygen functional groups on the GO surface can be easily tuned by the diffusion of these groups under an external electric field, which increases the reactivity of GOs towards ODH of propane. The chemically modified GOs are thus quite promising in the design of metal-free catalysis. PMID:22801590

  5. Structural Basis for the Active Site Inhibition Mechanism of Human Kidney-Type Glutaminase (KGA)

    PubMed Central

    Thangavelu, K.; Chong, Qing Yun; Low, Boon Chuan; Sivaraman, J.

    2014-01-01

    Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions. PMID:24451979

  6. Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase.

    PubMed

    Kiss, András L; Palló, Anna; Náray-Szabó, Gábor; Harmat, Veronika; Polgár, László

    2008-05-01

    It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k(cat)/K(m)) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme. PMID:18325786

  7. Bigger is better: honeybee colonies as distributed information-gathering systems

    PubMed Central

    Donaldson-Matasci, Matina C.; DeGrandi-Hoffman, Gloria; Dornhaus, Anna

    2015-01-01

    In collectively foraging groups, communication about food resources can play an important role in the organization of the group’s activity. For example, the honeybee dance communication system allows colonies to selectively allocate foragers among different floral resources according to their quality. Because larger groups can potentially collect more information than smaller groups, they might benefit more from communication because it allows them to integrate and use that information to coordinate forager activity. Larger groups might also benefit more from communication because it allows them to dominate high-value resources by recruiting large numbers of foragers. By manipulating both colony size and the ability to communicate location information in the dance, we show that larger colonies of honeybees benefit more from communication than do smaller colonies. In fact, colony size and dance communication worked together to improve foraging performance; the estimated net gain per foraging trip was highest in larger colonies with unimpaired communication. These colonies also had the earliest peaks in foraging activity, but not the highest ones. This suggests they may find and recruit to resources more quickly, but not more heavily. The benefits of communication we observed in larger colonies are thus likely a result of more effective informationgathering due to massive parallel search rather than increased competitive ability due to heavy recruitment. PMID:26213412

  8. The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism.

    PubMed

    Li, Jun; Machius, Mischa; Chuang, Jacinta L; Wynn, R Max; Chuang, David T

    2007-04-20

    A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction. PMID:17329260

  9. Novel active comb-shaped dry electrode for EEG measurement in hairy site.

    PubMed

    Huang, Yan-Jun; Wu, Chung-Yu; Wong, Alice May-Kuen; Lin, Bor-Shyh

    2015-01-01

    Electroencephalography (EEG) is an important biopotential, and has been widely applied in clinical applications. The conventional EEG electrode with conductive gels is usually used for measuring EEG. However, the use of conductive gel also encounters with the issue of drying and hardening. Recently, many dry EEG electrodes based on different conductive materials and techniques were proposed to solve the previous issue. However, measuring EEG in the hairy site is still a difficult challenge. In this study, a novel active comb-shaped dry electrode was proposed to measure EEG in hairy site. Different form other comb-shaped or spike-shaped dry electrodes, it can provide more excellent performance of avoiding the signal attenuation, phase distortion, and the reduction of common mode rejection ratio. Even under walking motion, it can effectively acquire EEG in hairy site. Finally, the experiments for alpha rhythm and steady-state visually evoked potential were also tested to validate the proposed electrode. PMID:25137719

  10. Targeting Large Kinase Active Site with Rigid, Bulky Octahedral Ruthenium Complexes

    SciTech Connect

    Maksimoska, Jasna; Feng, Li; Harms, Klaus; Yi, Chunling; Kissil, Joseph; Marmorstein, Ronen; Meggers, Eric

    2009-09-02

    A strategy for targeting protein kinases with large ATP-binding sites by using bulky and rigid octahedral ruthenium complexes as structural scaffolds is presented. A highly potent and selective GSK3 and Pim1 half-sandwich complex NP309 was successfully converted into a PAK1 inhibitor by making use of the large octahedral compounds {Lambda}-FL172 and {Lambda}-FL411 in which the cyclopentadienyl moiety of NP309 is replaced by a chloride and sterically demanding diimine ligands. A 1.65 {angstrom}cocrystal structure of PAK1 with {Lambda}-FL172 reveals how the large coordination sphere of the ruthenium complex matches the size of the active site and serves as a yardstick to discriminate between otherwise closely related binding sites.

  11. Jamestown: A Site Selection Exercise.

    ERIC Educational Resources Information Center

    Victory, James

    1982-01-01

    Describes a secondary, U.S. history simulation of the settlement of the Jamestown colony. Students are provided with a map and asked to decide where the fort and colony should be set up. They must state reasons for selecting a particular site and reasons for rejecting others. (AM)

  12. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. PMID:26291268

  13. Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-{kappa}B translocation

    SciTech Connect

    Jawan, Bruno; Kao, Y.-H.; Goto, Shigeru; Pan, M.-C.; Lin, Y.-C.; Hsu, L.-W.; Nakano, Toshiaki; Lai, C.-Y.; Sun, C.-K.; Cheng, Y.-F.; Tai, M.-H.

    2008-06-15

    Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 {mu}M after 48 h incubation. Pretreatment with 100 {mu}M PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-{alpha}, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and I{kappa}B{alpha}, as well as the nuclear translocation of NF-{kappa}B primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-{kappa}B nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers.

  14. Influence of cysteine 164 on active site structure in rat cysteine dioxygenase.

    PubMed

    Fellner, Matthias; Siakkou, Eleni; Faponle, Abayomi S; Tchesnokov, Egor P; de Visser, Sam P; Wilbanks, Sigurd M; Jameson, Guy N L

    2016-07-01

    Cysteine dioxygenase is a non-heme mononuclear iron enzyme with unique structural features, namely an intramolecular thioether cross-link between cysteine 93 and tyrosine 157, and a disulfide bond between substrate L-cysteine and cysteine 164 in the entrance channel to the active site. We investigated how these posttranslational modifications affect catalysis through a kinetic, crystallographic and computational study. The enzyme kinetics of a C164S variant are identical to WT, indicating that disulfide formation at C164 does not significantly impair access to the active site at physiological pH. However, at high pH, the cysteine-tyrosine cross-link formation is enhanced in C164S. This supports the view that disulfide formation at position 164 can limit access to the active site. The C164S variant yielded crystal structures of unusual clarity in both resting state and with cysteine bound. Both show that the iron in the cysteine-bound complex is a mixture of penta- and hexa-coordinate with a water molecule taking up the final site (60 % occupancy), which is where dioxygen is believed to coordinate during turnover. The serine also displays stronger hydrogen bond interactions to a water bound to the amine of the substrate cysteine. However, the interactions between cysteine and iron appear unchanged. DFT calculations support this and show that WT and C164S have similar binding energies for the water molecule in the final site. This variant therefore provides evidence that WT also exists in an equilibrium between penta- and hexa-coordinate forms and the presence of the sixth ligand does not strongly affect dioxygen binding. PMID:27193596

  15. Chronic exposure of a honey bee colony to 2.45 GHz continuous wave microwaves

    NASA Technical Reports Server (NTRS)

    Westerdahl, B. B.; Gary, N. E.

    1981-01-01

    A honey bee colony (Apis mellifera L.) was exposed 28 days to 2.45 GHz continuous wave microwaves at a power density (1 mW/sq cm) expected to be associated with rectennae in the solar power satellite power transmission system. Differences found between the control and microwave-treated colonies were not large, and were in the range of normal variation among similar colonies. Thus, there is an indication that microwave treatment had little, if any, effect on (1) flight and pollen foraging activity, (2) maintenance of internal colony temperature, (3) brood rearing activity, (4) food collection and storage, (5) colony weight, and (6) adult populations. Additional experiments are necessary before firm conclusions can be made.

  16. Chronic exposure of a honey bee colony to 2. 45 GHz continuous wave microwaves

    SciTech Connect

    Westerdahl, B.B.; Gary, N.E.

    1981-01-01

    A honey bee colony (Apis mellifera L.) was exposed 28 days to 2.45 GHz continuous wave microwaves at a power density (1 mW/sq cm) expected to be associated with rectennae in the solar power satellite power transmission system. Differences found between the control and microwave-treated colonies were not large, and were in the range of normal variation among similar colonies. Thus, there is an indication that microwave treatment had little, if any, effect on (1) flight and pollen foraging activity, (2) maintenance of internal colony temperature, (3) brood rearing activity, (4) food collection and storage, (5) colony weight, and (6) adult populations. Additional experiments are necessary before firm conclusions can be made.

  17. Molecular genetic evidence of formosan subterranean termite (Isoptera: Rhinotermitidae) colony survivorship after prolonged inundation.

    PubMed

    Owens, Carrie B; Su, Nan-Yao; Husseneder, Claudia; Riegel, Claudia; Brown, Kenneth S

    2012-04-01

    Levee breaches because of Hurricane Katrina in 2005 inundated 80% of the city of New Orleans, LA. Formosan subterranean termites were observed actively foraging within in-ground monitoring stations within months after this period of flooding. It was unknown if the activity could be attributed to preexisting colonies that survived inundation or to other colonies surviving flooding by being located at higher elevations readily invading these territories. Genotypic profiles of 17 termite colonies collected from eight inundated locations before flooding were compared with termite colonies after flooding from the same locations to determine Formosan subterranean termite survival after sustained flooding. Results indicate that 14 colonies were able to survive inundation for extended periods. PMID:22606822

  18. Ectoparasitism shortens the breeding season in a colonial bird.

    PubMed

    Brown, Charles R; Brown, Mary Bomberger

    2015-02-01

    When blood-feeding parasites increase seasonally, their deleterious effects may prevent some host species, especially those living in large groups where parasites are numerous, from reproducing later in the summer. Yet the role of parasites in regulating the length of a host's breeding season-and thus the host's opportunity for multiple brooding-has not been systematically investigated. The highly colonial cliff swallow (Petrochelidon pyrrhonota), a temperate-latitude migratory songbird in the western Great Plains, USA, typically has a relatively short (eight to nine week) breeding season, with birds rarely nesting late in the summer. Colonies at which ectoparasitic swallow bugs (Oeciacus vicarius) were experimentally removed by fumigation were over 45 times more likely to have birds undertake a second round of nesting than were colonies exposed to parasites. Late nesting approximately doubled the length of the breeding season, with some birds raising two broods. Over a 27 year period the percentage of birds engaging in late nesting each year increased at a colony site where parasites were removed annually. This trend could not be explained by changes in group size, climate or nesting phenology during the study. The results suggest that ectoparasitism shortens the cliff swallow's breeding season and probably prevents many individuals from multiple brooding. When this constraint is removed, selection may rapidly favour late nesting. PMID:26064606

  19. Structure/function correlations over binuclear non-heme iron active sites.

    PubMed

    Solomon, Edward I; Park, Kiyoung

    2016-09-01

    Binuclear non-heme iron enzymes activate O2 to perform diverse chemistries. Three different structural mechanisms of O2 binding to a coupled binuclear iron site have been identified utilizing variable-temperature, variable-field magnetic circular dichroism spectroscopy (VTVH MCD). For the μ-OH-bridged Fe(II)2 site in hemerythrin, O2 binds terminally to a five-coordinate Fe(II) center as hydroperoxide with the proton deriving from the μ-OH bridge and the second electron transferring through the resulting μ-oxo superexchange pathway from the second coordinatively saturated Fe(II) center in a proton-coupled electron transfer process. For carboxylate-only-bridged Fe(II)2 sites, O2 binding as a bridged peroxide requires both Fe(II) centers to be coordinatively unsaturated and has good frontier orbital overlap with the two orthogonal O2 π* orbitals to form peroxo-bridged Fe(III)2 intermediates. Alternatively, carboxylate-only-bridged Fe(II)2 sites with only a single open coordination position on an Fe(II) enable the one-electron formation of Fe(III)-O2 (-) or Fe(III)-NO(-) species. Finally, for the peroxo-bridged Fe(III)2 intermediates, further activation is necessary for their reactivities in one-electron reduction and electrophilic aromatic substitution, and a strategy consistent with existing spectral data is discussed. PMID:27369780

  20. Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

    PubMed Central

    Lee, Minho; Shin, Eunkyoung; Jeon, Che Ok; Cha, Chang-Jun; Han, Seung Hyun; Kim, Su-Jin; Lee, Sang-Won; Lee, Younghoon; Ha, Nam-Chul

    2011-01-01

    Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5′ termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. however, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart. Two substrate-bound tryptic peptides, 65HGFLPLK71—which includes amino acids previously implicated in substrate binding and catalysis—and 24LYDLDIESPGHEQK37—which includes the Q36 locus—were identified in wild-type enzyme complexes, whereas only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects. PMID:22186084

  1. The active sites in the heterogeneous Baeyer-Villiger oxidation of cyclopentanone by hydrotalcite catalysts

    NASA Astrophysics Data System (ADS)

    Ueno, Shinji; Ebitani, Kohki; Ookubo, Akira; Kaneda, Kiyotomi

    1997-11-01

    The active sites of hydrotalcites, [ Mg1-x2+Alx3+( OH) 2] x+[ Ax/nn-·m H 2O ] x-, A″ n-; CO 32-, Cl -, etc., were studied in the heterogeneous Baeyer-Villiger oxidation of cyclopentanone to δ-valerolactone with a combination oxidant system of molecular oxygen and benzaldehyde (O 2/benzaldehyde) in a 1,2-dichloroethane solvent. The hydrotalcites with different basicity were prepared by changing element ratios of Al 3+ to Mg 2+ in the Brucite-like layer and by changing anionic compounds (CO 32-, Cl -, and SO 42-) in the interlayer. The basicities of hydrotalcites were evaluated by measuring the calorimetric heats of benzoic acid adsorption and the zeta-potential of potassium chloride and by the indicator titration method. Yields of δ-valerolactone were almost proportional to the basicities of hydrotalcites, i.e., the heats of benzoic acid adsorption on hydrotalcites, which suggests that basic sites of hydrotalcites are active sites for the oxidation. Yields of δ-valerolactone were also dependent on the basicities of hydrotalcites using m-chloroperbenzoic acid ( m-CPBA) as an oxidant instead of O 2/benzaldehyde. Basic sites of hydrotalcites play an important role in the oxygen transfer from perbenzoic acid to ketone.

  2. Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity.

    PubMed

    Barinka, Cyril; Sácha, Pavel; Sklenár, Jan; Man, Petr; Bezouska, Karel; Slusher, Barbara S; Konvalinka, Jan

    2004-06-01

    Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question. PMID:15152093

  3. Barriers to physical activity in an on-site corporate fitness center.

    PubMed

    Schwetschenau, Heather M; O'Brien, William H; Cunningham, Christopher J L; Jex, Steve M

    2008-10-01

    Many corporations provide employees the option of participating in on-site fitness centers, but utilization rates are low. Perceived barriers to physical activity have been established as important correlates of physical activity, and recent research indicates that barriers may vary across settings. Work-site fitness centers may present unique barriers to participation, but there are currently no standardized measures that assess such barriers. Eighty-eight employees of a midwestern corporation completed a survey designed to identify and evaluate the extent to which barriers influence participation in an on-site corporate fitness center. Regression analyses revealed that external environmental barriers (e.g., inadequate exercise facilities) significantly accounted for not joining the fitness center, and for decreased duration of visits to the facility among members. Internal barriers (e.g., feeling embarrassed to exercise around coworkers) significantly accounted for frequency of fitness center visits among members. This corporate specific measure may lead to more effective interventions aimed to increase use of on-site corporate fitness centers. PMID:18837631

  4. Den site activity patterns of adult male and female swift foxes, Vulpes velox, in Northwestern Texas

    USGS Publications Warehouse

    Lemons, P.R.; Ballard, W.B.; Sullivan, R.M.; Sovada, M.A.

    2003-01-01

    Activity of Swift Foxes (Vulpes velox) at den sites was studied in northwestern Texas during pup rearing seasons in 2000 and 2001 to determine role of males in parental care. Twenty-four percent of radio-collared females with a potential to breed successfully raised pups to eight weeks of age. We intensively monitored presence and absence of male and female Swift Foxes at two den sites each year. Females were present >2.6 times more at den sites than males during the pup rearing season. Female and male Swift Foxes largely stayed at dens during diurnal hours and were active away from dens during nocturnal and crepuscular hours. Females and males spent 12.4% and 3.0% more time at dens before pups emerged, than after pups emerged, respectively. Following depredation of one male parent, the female spent 29% less time at the den site. Decrease in time spent at the den by the female following loss of her mate suggested that loss of one parent might severely impact recruitment of Swift Foxes. Our observations indicated that intense Coyote (Canis latrans) depredation may severely impact pup-rearing success as well as the parental care within Swift Fox family groups.

  5. Large zinc cation occupancy of octahedral sites in mechanically activated zinc ferrite powders

    SciTech Connect

    Oliver, S. A.; Harris, V. G.; Hamdeh, H. H.; Ho, J. C.

    2000-05-08

    The cation site occupancy of a mechanically activated nanocrystalline zinc ferrite powder was determined as (Zn{sub 0.55}{sup 2+}Fe{sub 0.18}{sup 3+}){sub tet}[Zr{sub 0.45}{sup 2+}Fe{sub 1.82}{sup 3+}]{sub oct}O{sub 4} through analysis of extended x-ray absorption fine structure measurements, showing a large redistribution of cations between sites compared to normal zinc ferrite samples. The overpopulation of cations in the octahedral sites was attributed to the ascendance in importance of the ionic radii over the crystal energy and bonding coordination in determining which interstitial sites are occupied in this structurally disordered powder. Slight changes are observed in the local atomic environment about the zinc cations, but not the iron cations, with respect to the spinel structure. The presence of Fe{sup 3+} on both sites is consistent with the measured room temperature magnetic properties. (c) 2000 American Institute of Physics.

  6. Evolution of a designed retro-aldolase leads to complete active site remodeling

    PubMed Central

    Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

    2013-01-01

    Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

  7. Twinning in fcc lattice creates low-coordinated catalytically active sites in porous gold.

    PubMed

    Krajčí, Marian; Kameoka, Satoshi; Tsai, An-Pang

    2016-08-28

    We describe a new mechanism for creation of catalytically active sites in porous gold. Samples of porous gold prepared by de-alloying Al2Au exhibit a clear correlation between the catalytic reactivity towards CO oxidation and structural defects in the fcc lattice of Au. We have found that on the stepped {211} surfaces quite common twin boundary defects in the bulk structure of porous gold can form long close-packed rows of atoms with the coordination number CN = 6. DFT calculations confirm that on these low-coordinated Au sites dioxygen chemisorbs and CO oxidation can proceed via the Langmuir-Hinshelwood mechanism with the activation energy of 37 kJ/mol or via the CO-OO intermediate with the energy barrier of 19 kJ/mol. The existence of the twins in porous gold is stabilized by the surface energy. PMID:27586937

  8. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates. PMID:27497172

  9. 13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites.

    PubMed

    McCullough, Christopher R; Pullela, Phani Kumar; Im, Sang-Choul; Waskell, Lucy; Sem, Daniel S

    2009-03-01

    The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a (13)CH(3)-reporter attached. This (13)C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site. PMID:19199046

  10. Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

    PubMed

    Zeymer, Cathleen; Werbeck, Nicolas D; Zimmermann, Sabine; Reinstein, Jochen; Hansen, D Flemming

    2016-09-12

    States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions. PMID:27534930

  11. The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.

    PubMed Central

    Rudd, M D; Izban, M G; Luse, D S

    1994-01-01

    RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active. Images PMID:8058756

  12. Extreme electric fields power catalysis in the active site of ketosteroid isomerase

    PubMed Central

    Fried, Stephen D.; Bagchi, Sayan; Boxer, Steven G.

    2015-01-01

    Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI’s rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme’s catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems. PMID:25525245

  13. Dynamics and Mechanism of Efficient DNA Repair Reviewed by Active-Site Mutants

    NASA Astrophysics Data System (ADS)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2010-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA via a photoreaction which includes a series of light-driven electron transfers between the two-electron-reduced flavin cofactor FADH^- and the dimer. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of several active-site residues. With femtosecond resolution, we observed the significant change in the forward electron transfer from the excited FADH^- to the dimer and the back electron transfer from the repaired thymines by mutation of E274A, R226A, R342A, N378S and N378C. We also found that the mutation of E274A accelerates the bond-breaking of the thymine dimer. The dynamics changes are consistent with the quantum yield study of these mutants. These results suggest that the active-site residues play a significant role, structurally and chemically, in the DNA repair photocycle.

  14. Synthesis of supported bimetallic nanoparticles with controlled size and composition distributions for active site elucidation

    SciTech Connect

    Hakim, Sikander H.; Sener, Canan; Alba Rubio, Ana C.; Gostanian, Thomas M.; O'neill, Brandon J; Ribeiro, Fabio H.; Miller, Jeffrey T.; Dumesic, James A

    2015-08-01

    Elucidation of active sites in supported bimetallic catalysts is complicated by the high level of dispersity in the nanoparticle size and composition that is inherent in conventional methods of catalyst preparation. We present a synthesis strategy that leads to highly dispersed, bimetallic nanoparticles with uniform particle size and composition by means of controlled surface reactions. We demonstrate the synthesis of three systems, RhMo, PtMo, and RhRe, consisting of a highly reducible metal with an oxophilic promoter. These catalysts are characterized by FTIR, CO chemisorption, STEM/EDS, TPR, and XAS analysis. The catalytic properties of these bimetallic nanoparticles were probed for the selective CO hydrogenolysis of (hydroxymethyl)tetrahydropyran to produce 1,6 hexanediol. Based on the characterization results and reactivity trends, the active sites in the hydrogenolysis reaction are identified to be small ensembles of the more noble metal (Rh, Pt) adjacent to highly reduced moieties of the more oxophilic metal (Mo, Re).

  15. Active site models for the Cu(A) site of peptidylglycine α-hydroxylating monooxygenase and dopamine β-monooxygenase.

    PubMed

    Kunishita, Atsushi; Ertem, Mehmed Z; Okubo, Yuri; Tano, Tetsuro; Sugimoto, Hideki; Ohkubo, Kei; Fujieda, Nobutaka; Fukuzumi, Shunichi; Cramer, Christopher J; Itoh, Shinobu

    2012-09-01

    A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated

  16. Mutation at a Strictly-Conserved, Active-Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity†

    PubMed Central

    Chen, Zhi-wei; Datta, Saumen; DuBois, J