1,581 research outputs found
Health consultation, W.R. Grace exfoliation facility : Phoenix, Maricopa County, Arizona
No full textabstract: The W.R. Grace facility in Phoenix, Arizona, received vermiculite concentrate from the Libby, Montana, vermiculite mine. W.R. Grace Company has owned and operated the Arizona site since 1964. In 1964 W.R. Grace purchased the company that had previously occupied the site and, following the relocation of its vermiculite exfoliation furnace from Glendale, Arizona, began processing vermiculite concentrate and marketing it under the Zonolite® brand. The objective of this health consultation is to evaluate exposure pathways and potential health effects in those persons who, between 1964 and 2002, may have been exposed to Libby asbestos as a result of vermiculite concentrate processing activities and waste materials from the W.R. Grace exfoliation facility in Phoenix.Under cooperative agreement with the Agency for Toxic Substances and Disease Registry.Includes bibliographical references (p. 22-24)
J. P. Bulduc with Helen and Don Ross
No full textJ.P. Bolduc, President and Chief Executive Officer of W.R. Grace and Co., poses with Mrs. Helen and Dr. Donald E. Ross on February 25, 1994 at the Dively Frontiers of Globalization Lecture event. Bolduc was the featured speaker.https://spiral.lynn.edu/dively-photos/1000/thumbnail.jp
Description of the KNMI Operational Wave Forecast Model GONO
No full textThe purpose of this report is to give a description of the GONO computercode, which is operational at KNMI for many years now. The program was developed by J.W. Sanders, and its deep water version is based on a Norwegian wave prediction model. built by C. Haug in the sixties. Shallow water effects are however important in the southern part of the North Sea. giving a limitation of the wave growth and causing important swell dissipation. A discussion of the shallow water effects. as present in GONO, is given by Sanders in Ref. 3. The computer code GONO is written in ALGOL60 and operational on the Burroughs 6700 of KNMI. The program GONO calculates wind speed. wind direction and sea energy at every grid point of the GONO grid (cf. Appendix A) and swell energy at a limited number of points only (we call these points swell points). GONO runs every six hours and it gives a 12 and 24 hours forecast as well as results based on analyzed weather maps. In the winter of '79-'80 the output of GONO was compared with measurements and the operational wave model of Bracknell (United Kingdom). (The latter GONO version differs a little bit from the version here described). The preliminary results of this comparison are given in Ref. 4. A reasonable agreement of significant wave height and low-frequency energy, as given by GONO, with the observations was found. The present version of GONO, with small modifications compared to the previous version, is operational since the end of February 1980. Essentially, the GONO model is based on two steps. First. the sea energy at every grid point is determined. To this end advection of energy is treated by means of a finite difference scheme whereas the growth of the wave energy is calculated by means of an empirical growth curve (assuming that the wave spectrum has a fixed form). The second step is the calculation of the swell. Of course, in principle swell can be treated likewise, but then one has to store swell energy (and its direction) at every time, at every grid point for every frequency band. Also. this finite difference scheme is rather crude for swell propagation. whereas. because of stability reasons. there is an upper bound for the propagation speed (in the present case the upper bound is given by 13.87 m/s). If one is only interested in swell information at particular points (swell points), it is tempting to use a ray technique. The advantage of this technique is that it is very accurate. Swell is determined in this fashion in GONO.GON
Phase transitions in single neurons and neural populations: Critical slowing, anesthesia, and sleep cycles
No full textThe firing of an action potential by a biological neuron represents a dramatic transition from small-scale linear stochastics (subthreshold voltage fluctuations) to gross-scale nonlinear dynamics (birth of a 1-ms voltage spike). In populations of neurons we see similar, but slower, switch-like there-and-back transitions between low-firing background states and high-firing activated states. These state transitions are controlled by varying levels of input current (single neuron), varying amounts of GABAergic drug (anesthesia), or varying concentrations of neuromodulators and neurotransmitters (natural sleep), and all occur within a milieu of unrelenting biological noise. By tracking the altering responsiveness of the excitable membrane to noisy stimulus, we can infer how close the neuronal system (single unit or entire population) is to switching threshold. We can quantify this “nearness to switching” in terms of the altering eigenvalue structure: the dominant eigenvalue approaches zero, leading to a growth in correlated, low-frequency power, with exaggerated responsiveness to small perturbations, the responses becoming larger and slower as the neural population approaches its critical point–-this is critical slowing. In this chapter we discuss phase-transition predictions for both single-neuron and neural-population models, comparing theory with laboratory and clinical measurement
Tungsten biochemistry of Pyrococcus furiosus
No full textTungsten is the heaviest element that exhibits biological activity (atomic number 74), when it is present in an enzyme. It is taken up by cells in the form of tungstate, and it is subsequently processed into an organic cofactor referred to as tungstopterin, which is found as active center in several enzymes. Pyrococcus furiosus is a hyperthermophilic archaeon that grows anaerobically at an optimal temperature of 100 ËšC, strictly dependent on the presence of tungstate. Over the last years, P. furiosus has become a model organism for hyperthermophiles, as many of its proteins have been the subject of research and its genome has been sequenced. Also regarding tungsten metabolism P. furiosus can be considered as a model: four tungsten containing aldehyde oxidoreductases were already characterized in some detail before the initiation of this study. In this thesis several aspects of tungsten metabolism in P. furiosus have been further explored: its tungstate transport system has been identified and characterized (WtpABC), studies on aspects of tungsten cofactor biosynthesis have been carried out and a new tungsten-containing aldehyde oxidoreductase (AOR), WOR5, has been purified and characterized.Applied Science
Biochemistry of Tungstoenzymes from Pyrococcus furiosus
No full textThe cell uses a variety of transition metals to provide greater catalytic diversity than could be achieved using only the functional groups of amino acids. The biochemistry of molybdenum and tungsten is unusual: they are the only 4d and 5d metal ions with established biological role(s). This thesis reports studies on the identification of tungstoenzymes using functional proteomics and on the physiological substrate, function and catalytic mechanism of one of the abundantly present tungstoenzymes of Pyrococcus furiosus; formaldehyde ferredoxin oxidoreductase (FOR). A new method of native-native two dimensional gel electrophoresis (2DGE) was developed in which no denaturants such as urea or SDS were applied (Chapter 2). Radioactively labeled tungsten (187WO42-) was added to the growth medium of P. furiosus to detect tungsten proteins for subsequent identification with mass spectrometry. The radiograms of gels containing radioactively labeled tungsten revealed six spots with tungsten-associated proteins. These spots were excised and analyzed by mass spectrometry. Two tungstoenzymes were identified as aldehyde ferredoxin oxidoreductase and formaldehyde ferredoxin oxidoreductase. No tungsten containing proteins could be identified from the other spots. A new tungsten containing oxidoreductase (WOR5) was discovered. It is the fifth and last member of the family of tungsten containing oxidoreductases from the hyperthermophilic archaeon P. furiosus. WOR5 was purified and characterized with EPR spectroscopy and electrochemistry (Chapter 3). It was found to be a homo-dimeric protein (subunit: 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. The enzyme has a broad substrate specificity with a high affinity for several substituted and non-substituted aliphatic and aromatic aldehydes with variable chain lengths, and it can use ferredoxin as electron acceptor in catalysis. The redox chemistry of the tungsten and iron-sulfur prosthetic groups in P. furiosus formaldehyde ferredoxin oxidoreductase (Chapter 4) and steady and pre-steady state kinetics (Chapter 5) for this enzyme were studied using formaldehyde as substrate and ferredoxin as electron acceptor. The intermediate, paramagnetic W(V) state could be trapped only by reduction of FOR with substrate, with consecutive one-electron intraprotein electron transfer to the single [4Fe-4S](2+;1+) cluster and partial comproportionation of the tungsten over W(IV, V, VI); this is a stable state in the absence of an external electron acceptor. Due to the very unfavorable hydratation equilibrium of the formaldehyde/methylene glycol couple no W(V) was found in dye mediated equilibrium redox titrations. The development of this intermediate was slow even at elevated temperatures and with a excess of substrate. The free formaldehyde, and not the methylene glycol, is the enzyme's substrate, implying that the KM for formaldehyde is three orders-of-magnitude less than the value previously reported in literature. The steady state kinetics of FOR is consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The determined KM of ferredoxin (14 μM) was an order of magnitude less than previously reported values, due to the fact that at high concentrations of substrate the enzyme is inhibited and denatured. Pre-steady state difference spectra revealed peak shifts and a lack of isosbestic points, an indication that several out-of-phase processes happen simultaneously in the first seconds of the reaction. The binding and the oxidation of the substrate are both fast processes. The release of the product and the electron shuffling over the tungsten and iron-sulfur center in the absence of an external electron acceptor are slower. Based on these results, in combination with results from previous EPR studies two alternatives for a catalytic redox cycle are proposed.Applied Science
Fungal Diversity and Cellulytic Activity in the Historic Huts, Ross Island, Antarctica
No full textThe goal of this study was to undertake a microbial investigation of the Historic Huts areas on Ross Island, to gain knowledge of the fungal biodiversity and biochemical framework, focusing on the wood degrading potential of these fungi at both psychrophilic (cold) and mesophilic (moderate) temperatures. Eight hundred and forty nine samples were collected from three Heroic Era Historic Huts of Antarctica, from a variety of substrates but predominantly structural wood. The huts, Discovery Hut at Hut Point, Terra Nova Hut at Cape Evans and Nimrod Hut at Cape Royds, are located on Ross Island and were all assembled in the early 20th century by the Heroic Era explorers to house the expeditions, stores and animals. These wooden huts were abandoned when the expeditions left. The introduction of wood and other organic material to a pristine environment along with the creation of a microclimate within the harsh Antarctica environment created interesting sites for studying fungal diversity, wood decay and fungal cellulase enzymes in an extreme environment. Each hut can be classified as offering different conditions and circumstances for fungal propagules. Of the three huts, Terra Nova Hut is the only hut where there are visible fungal blooms within the hut and it, with Discovery Hut, had the greatest number of samples that contained fungi compared to Nimrod Hut which had the least. Discovery Hut, at less than 500 metres from the United States McMurdo Station, is the most visited by scientist and base staff and has been the most demonstrably affected by human impact of the three huts due to its closeness to the research stations on Ross Island To ensure a full understanding of the fungal diversity of the Historic Hut sites, a variety of sampling techniques were used along with a variety of culture media. Two thousand and seventy six isolates consisting of 1177 filamentous fungi and 899 single celled microorganisms (yeast and bacteria) were isolated; all these cultures were frozen and now form the University of Waikato Antarctic Culture Collection. Five genera dominated the fungal isolates that were identified and these were Cladosporium, Geomyces, Cadophora, Penicillium and Thelebolus. The fungal diversity of these Historic Huts' communities is low but the members present are metabolically active, consistent with other microbial communities in the Antarctic. The Historic Huts and surroundings contain a diverse array of provision in the way of wood and supplies, which provide nutrient sources for fungal growth. Endemic organisms present in the soil could have been enriched by using the introduced nutrient sources as primary and/or second metabolic substrates. In addition, fungi could have been introduced with the wooden huts and supplies when they were brought to Antarctica by the Heroic Era explorers, or introduced in the subsequent years with visitors and conservation work conducted at the sites. These introduced organisms, though, would have had to adapt to the change in climate and conditions posed by the Antarctic in order to survive and be subsequently isolated in this study. A screen for carboxymethylcellulase (CMCase) activity was done on a selection of the fungal isolates as the first step to understand the cellulytic potential of the Antarctica fungal community inhabiting the huts. One hundred and six fungal isolates from a total of 404, that were screened were deemed to be CMCase positive, 27 fungal isolates were chosen for further study including quantifying the activity of extracellular endo-1,4-β-glucanase at psychrophilic and mesophilic incubation temperatures. All but one isolate could produce endo-1,4-β-glucanase activity at 4 C and many produced more endo-1,4-β-glucanase activity at 4 C than at 15 C. Cadophora malorum 182, Cadophora malorum 242, Penicillium roquefortii 405, Penicillium roquefortii 408, Geomyces sp. 711, Geomyces sp. 824 and Cladosporium oxysporium 805 were selected for in-depth study of growth characteristics including growth temperature preferences, growth on a variety of cellulose substrates, water activity, and carbon sources, the latter done by using a commercially available microtitre plate containing 95 carbon sources. All seven of the fungal isolates were classified as psychrotolerant and produced, when cultured at either 4 C or at 15 C, cellulase, protease, amylase, xylanase, and pectinase and mannanase enzyme activities. The range of water activity that the Antarctic Penicillium roquefortii isolates could grow at was distinctive when compared with food Penicillium roquefortii isolates. The utilisation of different carbon sources showed that like many studies of Antarctica organism they have a diverse range of enzymatic activity, but interestingly the activity does not differ greatly with incubation temperature with most carbon sources being used or not used at both incubation temperatures tested. Although it took longer for the fungi
to grow at the psychrophilic temperatures, the range of carbon sources they utilised was not reduced. The protein composition of the extracellular supernatants was visualised using various electrophoretic and staining techniques. The cellulase activity of the protein bands was visualised by cellulose-containing zymograms, which illustrated that the cellulase complex in all fungi tested was multi-enzyme and differed between species, isolates and temperatures of culturing. The cellulase activity of Cadophora malorum 182 was enriched by purification techniques including ion exchange chromatography and native preparative electrophoresis. The protein complex was not purified to homogeneity, but enriched for a mixture of proteins and the mixture was described as having the following properties; a temperature range of β-1,4-glucan cellobiohydrolase activity from 20 C to 80 C with the optimum activity seen at 60 C, β-1,4-glucan cellobiohydrolase activity that is stable at 4, 25 and 40 C for at least 24 hrs, lost at 50 C and 80 C within 24 hrs and 2 minutes respectively. Along with β-1,4-glucan cellobiohydrolase activity, the protein mixture contained Avicelase, CMCase, xylanase and mannanase activity. The thesis research showed that there was limited fungal diversity in the Historic Huts and artefacts (a total of five dominant genera were identified) but the fungi are actively growing and producing viable spores in the cold of Antarctica and producing the necessary enzymes for degradation of wood. Although the metabolism and growth rate is slower at psychrophilic temperatures, the fungal isolates studied as part of this thesis research could still function enzymatically at cold temperatures and this includes the degradation of wood as evidenced by in vitro wood decay studies examined by scanning electron microscopy where two isolates of one species demonstrated the ability to degrade wood. The cellulase complex of the investigated fungal isolate was multi-enzymed and although the components were not purified to homogeneity, an enriched mixture of proteins had enzyme activity and stability in a broad temperature range, and activity to a variety of cellulosic substrates.
This thesis research adds to the knowledge of the fungal biodiversity in the Antarctic and increases the understanding of the biochemical framework, participating in relation to wood decay potential of these Antarctic fungal isolates
Screening fungi isolated from historic Discovery Hut on Ross Island, Antarctica for cellulose degradation
No full textTo survive in Antarctica, early explorers of Antarctica's Heroic Age erected wooden buildings and brought in large quantities of supplies. The introduction of wood and other organic materials may have provided new nutrient sources for fungi that were indigenous to Antarctica or were brought in with the materials. From 30 samples taken from Discovery Hut, 156 filamentous fungi were isolated on selective media. Of these, 108 were screened for hydrolytic activity on carboxymethyl cellulose, of which 29 demonstrated activities. Endo-1, 4-β-glucanase activity was confirmed in the extracellular supernatant from seven isolates when grown at 4°C, and also when they were grown at 15°C. Cladosporium oxysporum and Geomyces sp. were shown to grow on a variety of synthetic cellulose substrates and to use cellulose as a nutrient source at temperate and cold temperatures. The research findings from the present study demonstrate that Antarctic filamentous fungi isolated from a variety of substrates (wood, straw, and food stuffs) are capable of cellulose degradation and can grow well at low temperatures
Structural and functional studies of the iron storage protein ferritin from Pyrococcus furiosus
No full textThis research focuses on the iron storage protein ferritin. Ferritin is a protein involved in iron homeostasis by storing Fe(II) excess in the form of an Fe(III) mineral core in the presence of oxygen and by releasing iron during iron deficiency. Ferritins are vital for human health. Their malfunction may lead among other diseases to anemia, iron overload, Parkinson or Alzheimer. In addition to its medical significance, there are industrial applications of ferritin such as in nanotechnology, catalysis and environmental cleaning. The research of this study focused on ferritin from hyperthermophile, archaeon and anaerobe Pyrococcus furiosus and involved both the fundamental biochemistry of ferritin and ferritin's possible application. The results include the development of a highly efficient overproduction system of P. furiosus ferritin, the determination of ferritin's redox chemistry that gave insight into ferritin's possible physiological redox partners, and determination of P. furiosus ferritin's crystal structure. Additionally, ferritin has been used for the production of carbon nanotubes and a method to increase the yield of carbon nanotubes was developed and presented.Applied Science
Redox biochemistry of Pyrococcus furiosus: Fundamental and applied aspects
No full textIn Pyrococcus furiosus (Pfu) ferredoxin (Fd) replaces NAD+ for carrying reducing equivalent to the oxidative phosphorylation machinery, and it also takes part in the regeneration of NADPH, which is necessary for biosynthetic pathways. Therefore, it plays a central role in various metabolic pathways of the organism. PfuFd is one of the most extensively studied ferredoxins, however, some fundamental aspects remain elusive. This thesis work focuses on some of the unresolved features of the protein. Initially PfuFd was isolated as a dimer of 12-13 kDa, however, all the subsequent studies described the protein as a monomer without any experimental evidence. All (putative) natural electron-transfer partners of PfuFd are redox enzymes catalyzing two-electron reactions while ferredoxin is a one-electron carrier. It is possible that ferredoxin may interact with the redox partner enzymes in the dimeric form. Therefore, it is important to resolve the dilemma regarding the oligomeric state of the protein (chapter 2). The intra-cellular ionic strength of P. furiosus was determined to be ca. 350 mM, at which ferredoxin occurs predominantly in the dimeric form. Transition from the dimeric to monomeric form is observed at a salt concentration higher than 350 mM. We hypothesize that ferrdoxin is a dimer in vivo. PfuFd demonstrates conservation of the minimal domain containing the cluster coordinating consensus sequence. However, the cluster is coordinated by three cysteines and one aspartate residue instead of by four cysteines. In addition, two additional cysteines are present which raises the possibility of a disulfide bond formation between them. In some earlier studies the formation of a disulfide bond was demonstrated under aerobic condition and a possible contribution of the disulfide bond to the redox chemistry of the protein has been described. However, formation of a disulfide bond under the strongly reducing intracellular condition of an anaerobic organism like P. furiosus is not understandable. We demonstrate (in chapter 3) that these additional cysteines do not form a disulfide bond under intra-cellular mimicking conditions. Observing the effects of site directed mutagenesis, we attribute to the free cysteines an important structural role in the hyperthermostability of the protein. Ferredoxin, cytochrome c, rubredoxin, etc. are small electron-transfer protein and they possess well characterised metal cofactors. Electrochemistry has been successfully applied in studying the intricate properties of these electron-transfer proteins mainly in solution. However, solution voltammetry suffers from various problems like mass transport / diffusion limitation, requirement of promoters, which can be avoided by immobilizing the protein on the electrode surface. A stable and functional PfuFd immobilized gold electrode has been developed, which shows a similar electrochemical response as in the solution voltammetry. The transition between the dimeric and monomeric state on the electrode is observed in an atomic force microscopic (AFM) setup. X-ray photoelectron spectrocopy (XPS) confirms the formation of gold-thiol bonds. We find the ferredoxin electrode to be a useful tool for studying the catalytic mechanisms of the associated redox enzymes from P. furiosus. We have thus studied two redox enzymes, glyceraldehyde-3-phosphate oxidoreductase (GAPOR) and aldehyde oxidoreductase (AOR), which are involved in ferredoxin-mediated redox processes in the organism. Addition of these enzymes at room temperature results in complex formation between the electrode-bound ferredoxin and the enzyme. At 60 °C a catalytic wave appears upon addition of the substrate, glyceraldehyde-3-phosphate to the Fd-GAPOR complex. In the case of AOR at 80 °C reversible oxidation of crotonadehyde to crotonic acid and vice versa was observed. This work opens the way for the application of Fd electrodes in achieving controlled reduction of carboxylic acids on a preparative scale.Applied Science
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