Studies on the unfolding and refolding of oligomeric proteins

The unfolding and refolding of a number of oligomeric enzymes have been studied. These were: fumarase from pig heart, the NAD+ -dependent isocitrate dehydrogenase from yeast, the citrate synthases from pig hean, Acinetobacter anitratum and Thermoplasma acidophilum and the chaperone protein GroEL from Escherichia coli. In each case the unfolding by guanidinium chloride (GdnHCI) was monitored by enzyme activity (to detect possible perturbations at the active site), protein fluorescence (to detect changes in tertiary structure) and far U.v. circular dichroism (to detect changes in protein secondary structure). In general the losses in secondary and tertiary structure were found to run broadly in parallel, whereas the enzyme activity was lost at much lower concentrations of GdnHCl. This sensitivity to mild, denaturing conditions may reflect the greater flexibility of the active site compared with the molecule as a whole. Interestingly) the bacterial citrate synthases were activated in the presence of low concentrations of GdnHCl. Following denaturation) refolding was initiated by lowering the concentration of GdnHCI by dilution or dialysis. Only the dimeric citrate synthases (from pig heart and Thermoplasma acidophilum) could be reactivated to a moderate extent using the dilution procedure; less than 5% reactivation was observed for the other enzymes. In the cases of fumarase, NAD+ -dependent isocitrate dehydrogenase and the dimeric citrate synthases the degrees of reactivation following dialysis were significantly greater (approximately 50-75% of the native enzymes) than those obtained following the dilution procedure. Factors such as protein concentration and the inclusion of dithiothreitol in the dialysis or dilution buffer were found to influence significantly the extent of reactivation. The greater yield of reactivation of unfolded protein using the dialysis procedure probably reflects the ability of the enzyme to make the correct structural adjustments between intermediates when the concentration of GdnHCI is lowered gradually. In the case of Thermoplasma acidophilum the recovery of citrate synthase activity was much greater at 20 ·C than at 55 ·C (the optimal temperature for growth of this organism). This has implications for the folding process in vivo under the extreme growth conditions of thermophiles and possibly other extremophiles. The hexameric citrate synthase from Acinetobacter anitratum and the tetradecameric chaperonin, GroEL could not be reactivated following denaturation. Far u.v. circular dichroism measurements on GroEL indicated that the native secondary structure of this protein was regained to a large extent. In vivo a number of the proteins studied (fumarase and citrate synthase from pig hean and yeast NAD+ -dependent isocitrate dehydrogenase)are translocated into mitochondria as precursors in a non-native state prior to processing, folding and assembly. The lack of complete refolding of the proteins studied in this work points to the existence of specialised mechanisms in vivo to promote efficient folding. Chaperone proteins have been implicated in the assistance of protein folding in vivo. Intriguingly. the studies on the inefficient refolding of the chaperonin GroEL support the proposal that this protein may fold in vivo by way of a "self chaperoning" mechanism

Late Victorian Sexuality and Spiritualism: The Place of the Paranormal in Queer Erotic Partnerships

This dissertation argues that fin de siecle navigations of emerging categories of sexual identity were partially expressed in literary representations of supernatural connections, transformations, events, and practices. I build on Sexuality Studies and Queer Theory foundations by such scholars as Foucault, Halberstam, and Warner along with New Historicist scholarship about spiritualism in the nineteenth century to examine the connections between aberrant erotic desires and alternative forms of spirituality. Through readings of the anonymous Teleny (1893), Wilde\u27s The Picture of Dorian Gray, Richard Marsh\u27s The Beetle , and the poetry of Michael Field, I assert that queer sexual identities and acts, unspeakable, unfixed, and nebulous in the late Victorian years, were supported, represented, and navigated in these works through the use of spiritualism of various kinds. I conclude my project by examining how the Neo-Victorian Showtime series Penny Dreadful (2014-2016) reshapes Victorian practices into a new resource for the modern LGBTQ+ community, demonstrating the continuing importance of spiritualism and sexual desire in non-traditional identity and relationship building

A neutral protease of the neutrophil surface : role in the proteolysis of C-reactive protein and fibrinogen

Both of the acute phase reactants, C-reactive protein and fibrinogen, as well as neutrophils have been shown to accumulate at sites of tissue injury or inflammation. The association of C-reactive protein with neutrophils and the concomitant degradation of this ligand by a phorbol 12-myristate 13 acetateactivatable membrane-associated neutral protease has been shown in previous studies. Degradation of C-reactive protein by the neutrophil protease was shown to result in peptides with an ability to modulate various immune functions of the neutrophil. The aim of this study has been to investigate specific characteristics of the protease, with respect to cellular distribution and molecular size. The ability of this neutrophil membrane-associated protease to degrade the acute phase protein, fibrinogen was investigated. The mechanism of degradation of both C-reactive protein and fibrinogen during their association with the neutrophil was also examined. The neutrophil protease, capable of degrading C-reactive protein, was also associated with the cytoskeleton and was proposed to be a submembrane protease localised at sites of attachment of the membrane with the cytoskeleton. The protease was found to have a molecular mass of approximately 600 kDa which, on sodium dodecyl sulphate polyacrylamide gel electrophoresis, separated into four bands which migrated to molecular mass values of 209 kDa, 316 kDa, 398 kDa and 501 kDa. This protease also possessed fibrinogenolytic activity. The fibrinogen degradation products generated by this neutrophil membrane-associated protease were distinct from the products generated by the fibrinogenolytic systems of plasmin, human neutrophil elastase and neutrophil lysosomal enzymes and were unclottable through cleavage of the Aα chain from the N-terminus and the Bβ and γ chains from the C-terminus. N-terminal cleavage of the Aα chain by the neutrophil membrane-associated protease generated the Aα1-21 peptide, previously regarded as a unique consequence of elastase activity. Degradation of C-reactive protein and fibrinogen occurred as a result of their interaction with the neutrophil near to the CD11c integrin receptor. This interaction resulted in the egress of proteolytic activity into the extracellular medium. The fibrinogen products generated outside the cell associated with the neutrophil via the β₂ integrin receptors and the IgG Fc receptor. The interaction of the Creactive protein degradation products with the neutrophil could not be determined. Both C-reactive protein and fibrinogen are degraded by non-stimulated neutrophils but activation with phorbol 12- myristate 13 acetate resulted in maximum degradation This upregulation of activity was achieved through activation of H7 and trifluoperazine inhibitable cellular kinases and changes in microfilament assembly. The generation of non-clottable fibrinogen together with possible modulation of neutrophil receptormediated functions by the fibringen degradation products as well as the knowledge that the neutrophil protease generates C-reactive protein peptides with immunomodulatory activity implicates this neutrophil membrane-associated protease in the modulation of various inflammatory processes

Exploring the Impact of Place-Conscious Pedagogy on Science Education

Our research explores the impact of place-conscious pedagogy on science education in the context of small seaport and prairie communities in Canada. Place-conscious pedagogy offers hands-on opportunities for students to become active participants in understanding and applying scientific knowledge and methods in local environmental contexts. Our study is guided by two interrelated questions: what practices do place-conscious teachers offer science students and how do these teachers understand the impact of place-conscious pedagogy on science education? We explore these questions through in-depth teacher interviews and photographic documentation of related classroom practices and outcomes. This information is used to generate case studies that further our understanding of place-conscious pedagogy as a practical means of enhancing science education—a core curricular expectation—and as a support for fostering social transformation and environmental sustainability. Our presentation focuses on two such cases. One case enlisted high school students in a population count of the invasive green crab. The second case engaged elementary students in field observations of owls. A theme we explored in our case studies was the relationship between place-conscious pedagogy and transformative student agency. Our research indicates that when students actively participate in understanding and shaping the world around them, they learn to recognize themselves as transformative agents of change (Kelly & Pelech, 2019; Pelech & Kelly, 2020). These cases offer practical illustrations of teaching that can enhance prospects for making science meaningful to students and that promote long-term environmental and social transformation

Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1

Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen- or voltage-sensing (LOV) domains within their N-terminus, with each binding one molecule of flavin mononucleotide (FMN) as a UV/blue light absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A′α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2-core and its flanking helical segments can uncouple this mode of light regulation. Yet, the impact of these mutations on phot function in Arabidopsis has not been explored. Here, we report that histidine substitution of Arg-472 located within the A′α-helix of Arabidopsis phot1 to histidine results in constitutively activates kinas activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo, but was unable to initiate phot1 signaling in the absence of light. Instead, we found that the phot1 R472H mutant is poorly functional under low-light conditions, but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant by in part reducing its stability, whereas the activity the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation

Automatic translation among spoken languages

The Machine Aided Voice Translation (MAVT) system was developed in response to the shortage of experienced military field interrogators with both foreign language proficiency and interrogation skills. Combining speech recognition, machine translation, and speech generation technologies, the MAVT accepts an interrogator's spoken English question and translates it into spoken Spanish. The spoken Spanish response of the potential informant can then be translated into spoken English. Potential military and civilian applications for automatic spoken language translation technology are discussed in this paper

Experience with Carbon Taxes and Greenhouse Gas Emissions Trading Systems

Carbon taxes and emissions trading systems (ETSs) to limit emissions of greenhouse gases (GHGs) are increasingly common. At the end of 2015, 17 GHG ETSs were operational in 55 jurisdictions, and 18 jurisdictions collected at least one carbon tax. This paper assesses the performance of carbon taxes and ETSs with respect to environmental effectiveness (reduction of emissions regulated by the instrument), cost-effectiveness (marginal abatement cost), economic efficiency, public finance, and administrative issues. Data on emissions subject to carbon taxes are rarely reported. We estimate the taxed emissions for 17 taxes in 12 jurisdictions from 1991 through the end of 2015. All 17 taxes have reduced emissions relative to business-as-usual. Six of the jurisdictions actually reduced emissions, although in at least three of those jurisdictions the reductions appear to be due to other policies. The small sizes of reduction in almost all 17 cases are partially due to the low tax rates; the modest and uncertain changes in tax rates over time; and the limited response of taxed sources, such as fossil fuels, to price changes. Actual emissions declined for at least six of 10 ETSs. Other policies and developments, such as the 2009 recession, contributed to the reductions, but estimates of the share of the reduction attributable to the instrument are rare. All of the ETSs have accumulated banks of surplus allowances and most have implemented measures to reduce these banks. On average, the marginal cost of compliance is substantially lower for ETSs than carbon taxes. ETS experience has been shared bilaterally and via dedicated institutions. As a result, most ETSs have increased the share of allowances auctioned; adopted declining emissions caps; specified future caps and floor prices several years into the future; shifted to benchmarking for free allowance allocations to emissions-intensive, trade-exposed (EITE) sources; reduced accessibility to foreign offset credits; and established market stability reserves. By contrast, there is little evidence of shared learning and virtually no change to the design of carbon taxes. We found no jurisdiction that routinely tracks the taxed emissions. Very few jurisdictions regularly assess the effectiveness of the tax in achieving emission reductions. Additionally, adjustments to the tax rate often are unpredictable after an introductory period of three to five years. Both instruments reduce emissions, but ETSs have performed better than carbon taxes on the principal criteria of environmental effectiveness and cost-effectiveness. Many jurisdictions have implemented both a carbon tax and a GHG ETS, and every jurisdiction that has adopted either instrument has also implemented other policies. More research is needed to improve the design of both instruments and their interaction with non-market-based carbon policies because the use of multiple instruments produces complex interactive and distributional effects. While economically inefficient, market-based policies should be supplemented by non-market-based policies to ensure sustained political support