High Resolution X-ray and Neutron Crystallographic Studies of \u3cem\u3eEscherichia coli\u3c/em\u3e Dihydrofolate Reductase

Dihydrofolate Reductases (DHFRs) have been identified in nearly every proteome and are essential for most biosynthetic pathways involving one-carbon transfer reactions due to their recycling of tetrahydrofolate (THF). They catalyze the NADPH-dependent reduction of dihydrofolate (DHF), producing THF. Inhibition of DHFR ultimately depletes cellular pools of THF; causing a reduced supply of thymine nucleotides for DNA synthesis, resulting in genomic instability and cell death. Therefore, DHFRs remain important drug targets in antimicrobial and chemotherapeutic treatments. Despite exhaustive investigation of E. coli chromosomal DHFR, controversy persists over the dynamics of regulatory loops (the Met20, the βF-βG, and the βG-βH) and the nature of the interaction between methotrexate (MTX), a tight-binding anti-cancer drug, and Asp 27, the only ionizable residue in the active site. Also of importance is the ionization state of Asp 27 in the apoenzyme and other complexes. Hydrogen atoms (H) likely play a critical role in DHFR ligand binding and catalysis, yet are difficult to directly visualize. High resolution X-ray and neutron crystallography have been utilized in this dissertation to provide accurate positions of H within the DHFR active site and to probe dynamics of the enzyme. The ultrahigh resolution X-ray structures of DHFR/MTX (1.0Å; chapter 4), apo DHFR (1.05Å), and DHFR/MTX/NADPH (1.4Å; both chapter 5) have been solved. Novel features were observed in the electron density maps, including the ability to model the Met20 loop in the apoenzyme as closed (reported disordered previously) and alternate side chain conformations in all the structures. The high data-to-parameter ratio of the apoenzyme and the MTX data sets allowed anisotropic B-factor refinement and full-matrix refinement to calculate carboxylate bond lengths and estimates of their deviations. The apoenzyme has highly different bond lengths for its Asp 27 carboxylate, thus, it is neutral at physiological pH. The carboxylate bond lengths of the Asp 27 in both the monomers of the asymmetric unit of the DHFR/MTX crystal are nearly equal, suggesting it is charged at physiological pH. If H is substituted for deuterium (D), neutrons are especially powerful probes due to D’s strong positive scattering length. To assign protonation states to the MTX and the Asp 27 by the direct identification of D, a neutron structure has been solved to 2.2Å resolution from nearly 80% complete data collected on a 0.3mm3 crystal (chapter 4). Prerequisite to the neutron experiment was the growth and D2O-soaking of large-volume crystals (chapter 3). The DHFR/MTX cocrystal possesses the largest primitive unit cell and is the smallest D2O-soaked crystal used successfully in a neutron diffraction experiment. This is the 11th novel protein ever to be solved by neutron crystallography (the 16th total structure). Nearly 2/3 of the amide backbone has undergone H/D exchange, an indicator of protein dynamics. However, monomer B, where the Met20 loop is closed, is ~10% more exchanged than monomer A, where the Met20 loop is partially occluded. Based on results from D occupancy refinement and analysis of the neutron maps, it is concluded that the MTX N1 is protonated when bound to DHFR. Paired with the X-ray data, this is new strong evidence that the Asp 27·MTX interaction is ionic in nature. To increase the signal-to-noise ratio in future neutron experiments, perdeuterated protein has been produced and its D enrichment measured by mass spectrometry. X-ray data (to 1.2Å) has now been collected on a perdeuterated DHFR/MTX cocrystal and it is isomorphous to the native cocrystals (chapter 3)

An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca(2+) blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca(2+). The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca(2+) coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca(2+)chelation. Computational analysis revealed that Ca(2+)-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K(+) into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore

Enhancing hole mobility in III-V semiconductors

Transistors based on III-V semiconductor materials have been used for a variety of analog and high frequency applications driven by the high electron mobilities in III-V materials. On the other hand, the hole mobility in III-V materials has always lagged compared to group-IV semiconductors such as silicon and germanium. In this paper we explore the used of strain and heterostructure design guided by bandstructure modeling to enhance the hole mobility in III-V materials. Parameters such as strain, valence band offset, effective masses and splitting between the light and heavy hole bands that are important for optimizing hole transport are measured quantitatively using various experimental techniques. A peak Hall mobility for the holes of 960cm2/Vs is demonstrated and the high hole mobility is maintained even at high sheet charge.Comment: 18 pages, 21 figure

A Neptune-mass Planet Orbiting the Nearby G Dwarf HD16417

Precision Doppler measurements from an intensive 48 night "Rocky Planet Search" observing campaign on the Anglo-Australian Telescope (AAT) have revealed the presence of a low-mass exoplanet orbiting the G1 dwarf HD16417. Subsequent Doppler observations with the AAT, as well as independent observations obtained by the Keck Planet Search, have confirmed this initial detection and refine the orbital parameters to period 17.24+/-0.01 d, eccentricity 0.20+/-0.09, orbital semi-major axis 0.14+/-0.01 AU and minimum planet mass 22.1+/-2.0 Mearth. HD 16417 raises the number of published exoplanets with minimum masses of less than 25 Mearth to eighteen. Interestingly, the distribution of detected sub-25 Mearth planets over the spectral types G, K and M is almost uniform. The detection of HD 16417b by an intensive observing campaign clearly demonstrates the need for extended and contiguous observing campaigns when aiming to detect low-amplitude Doppler planets in short period orbits. Perhaps most critically it demonstrates that the search for low-mass Doppler planets will eventually require these traditional "bright-time" projects to extend throughout dark lunations.Comment: To appear in Ap

A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2

Summary The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors

In Search of a Trade Mark: Search Practices and Bureaucratic Poetics

Trade marks have been understood as quintessential ‘bureaucratic properties’. This article suggests that the making of trade marks has been historically influenced by bureaucratic practices of search and classification, which in turn were affected by the possibilities and limits of spatial organisation and technological means of access and storage. It shows how the organisation of access and retrieval did not only condition the possibility of conceiving new trade marks, but also served to delineate their intangible proprietary boundaries. Thereby they framed the very meaning of a trade mark. By advancing a historical analysis that is sensitive to shifts, both in actual materiality and in the administrative routines of trade mark law, the article highlights the legal form of trade mark as inherently social and materially shaped. We propose a historical understanding of trade mark law that regards legal practice and bureaucratic routines as being co-constitutive of the very legal object itself

Agribusiness Sheep Updates - 2004 part 2

Precision Pastures Using Species Diversity to Improve Pasture Performance Anyou Liu and Clinton Revell, Department of Agriculture, Western Australia New Annual Pasture Legumes for Sheep Graziers Phil Nichols, Angelo Loi, Brad Nutt and Darryl McClements Department of Agriculture Western Australia Pastures from Space – Can Satellite Estimates of Pasture Growth Rate be used to Increase Farm Profit? Lucy Anderton, Stephen Gherardi and Chris Oldham Department of Agriculture Western Australia Summer-active Perennial Grasses for Profitable Sheep Production Paul Sanford and John Gladman, Department of Agriculture, Western Australia Pastures From Space – Validation Of Predictions Of Pasture Growth Rates DONALD, G.E.A, EDIRISINGHE, A.A, HENRY, D.A.A, MATA, G.A, GHERARDI, S.G.B, OLDHAM, C.M.B, GITTINS, S.P.B AND SMITH, R. C. G.C ACSIRO, Livestock Industries, PMB 5, Wembley, WA, 6913. BDepartment of Agriculture Western Australia, Bentley, WA, 6983. C Department of Land Information Western Australia, Floreat, WA, 6214. Production and Management of Biserrula Pasture - Managing the Risk of Photosensitivity Dr Clinton Revell and Roy Butler, Department of Agriculture Western Australia Meat Quality of Sheep Grazed on a Saltbush-based Pasture Kelly Pearce1,2, David Masters1, David Pethick2, 1 CSIRO LIVESTOCK INDUSTRIES, WEMBLEY, WA 2 SCHOOL OF VETERINARY AND BIOMEDICAL SCIENCE, MURDOCH UNIVERSITY, MURDOCH, WA Precision Sheep Lifetime Wool – Carryover Effects on Subsequent Reproduction of the Ewe Flock Chris Oldham, Department of Agriculture Western Australia Andrew Thompson, Primary Industries Research Victoria (PIRVic), Dept of Primary Industries, Hamilton, Vic Ewe Productivity Trials - a Linked Analysis Ken Hart, Johan Greeff, Department of Agriculture Western Australia, Beth Paganoni, School of Animal Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia. Grain Finishing Systems For Prime Lambs Rachel Kirby, Matt Ryan, Kira Buttler, Department of Agriculture, Western Australia The Effects of Nutrition and Genotype on the Growth and Development, Muscle Biochemistry and Consumer Response to Lamb Meat David Pethick, Department of Veterinary Science, Murdoch University, WA, Roger Heggarty and David Hopkins, New South Wales Agriculture ‘Lifetime Wool’ - Effects of Nutrition During Pregnancy and Lactation on Mortality of Progeny to Hogget Shearing Samantha Giles, Beth Paganoni and Tom Plaisted, Department of Agriculture Western Australia, Mark Ferguson and Darren Gordon, Primary Industries Research Victoria (PIRVic), Dept of Primary Industries, Hamilton, Vic Lifetime Wool - Target Liveweights for the Ewe Flock J. Young, Farming Systems Analysis Service, Kojonup, C. Oldham, Department of Agriculture Western Australia, A. Thompson, Primary Industries Research Victoria (PIRVic), Hamilton, VIC Lifetime Wool - Effects of Nutrition During Pregnancy and Lactation on the Growth and Wool Production of their Progeny at Hogget Shearing B. Paganoni, University of Western Australia, Nedlands WA, C. Oldham, Department of Agriculture Western Australia, M. Ferguson, A. Thompson, Primary Industries Research Victoria (PIRVic), Hamilton, VIC RFID Technology – Esperance Experiences Sandra Brown, Department of Agriculture Western Australia The Role of Radio Frequency Identification (RFID) Technology in Prime Lamb Production - a Case Study. Ian McFarland, Department of Agriculture, Western Australia. John Archer, Producer, Narrogin, Western Australia Win with Twins from Merinos John Milton, Rob Davidson, Graeme Martin and David Lindsay The University of Western Australia Precision Sheep Need Precision Wool Harvesters Jonathan England, Castle Carrock Merinos, Kingston SE, South Australia Business EBVs and Indexes – Genetic Tools for your Toolbox Sandra Brown, Department of Agriculture Western Australia Green Feed Budget Paddock Calculator Mandy Curnow, Department of Agriculture Western Australia Minimising the Impact of Drought - Evaluating Flock Recovery Options using the ImPack Model Karina P. Wood, Ashley K. White, B. Lloyd Davies, Paul M. Carberry, NSW Department of Primary Industries (NSW DPI), Lifetime Wool - Modifying GrazFeed® for WA Mike Hyder, Department of Agriculture Western Australia , Mike Freer, CSIRO Plant Industry, Canberra, A.C.T. , Andrew van Burgel, and Kazue Tanaka, Department of Agriculture Western Australia Profile Calculator – A Way to Manage Fibre Diameter Throughout the Year to Maximise Returns Andrew Peterson, Department of Agriculture, Western Australia Pasture Watch - a Farmer Friendly Tool for Downloading and Analysing Pastures from Space Data Roger Wiese,Fairport Technologies International, South Perth, WA, Stephen Gherardi, BDepartment of Agriculture Western Australia, Gonzalo Mata, CCSIRO, Livestock Industries, Wembley, Western Australia, and Chris Oldham, Department of Agriculture Western Australia Sy Sheep Cropping Systems An Analysis of a Cropping System Containing Sheep in a Low Rainfall Livestock System. Evan Burt, Amanda Miller, Anne Bennett, Department of Agriculture, Western Australia Lucerne-based Pasture for the Central Wheatbelt – is it Good Economics? Felicity FluggeA, Amir AbadiA,B and Perry DollingA,B,A CRC for Plant-based Management of Dryland Salinity: BDept. of Agriculture, WA Sheep and Biserrula can Control Annual Ryegrass Dean Thomas, John Milton, Mike Ewing and David Lindsay, The University of WA, Clinton Revell, Department of Agriculture, Western Australia Sustainable Management Pasture Utilisation, Fleece Weight and Weaning Rate are Integral to the Profitability of Dohnes and SAMMs. Emma Kopke,Department of Agriculture Western Australia, John Young, Farming Systems Analysis Service Environmental Impact of Sheep Confinement Feeding Systems E A Dowling and E K Crossley, Department of Agriculture, Western Australia Smart Grazing Management for Production and Environmental Outcomes Dr Brien E (Ben) Norton, Centre for the Management of Arid Environments, Curtin University of Technology, WA Common Causes of Plant Poisoning in the Eastern Wheatbelt of Western Australia. Roy Butler, Department of Agriculture, Western Australia Selecting Sheep for Resistance to Worms and Production Trait Responses John Karlsson, Johan Greeff, Department of Agriculture, Western Australia, Geoff Pollott, Imperial College, London UK Production and Water Use of Lucerne and French Serradella in Four Soil Types, Diana Fedorenko1,4, Darryl McClements2,4 and Robert Beard3,4, 12Department of Agriculture, Western Australia; 3Farmer, Meckering; 4CRC for Plant-based Management of Dryland Salinity. Worm Burdens in Sheep at Slaughter Brown Besier, Department of Agriculture Western Australia, Una Ryan, Caroline Bath, Murdoch Universit

Narcissism and the strategic pursuit of short-term mating : universal links across 11 world regions of the International Sexuality Description Project-2.

Previous studies have documented links between sub-clinical narcissism and the active pursuit of short-term mating strategies (e.g., unrestricted sociosexuality, marital infidelity, mate poaching). Nearly all of these investigations have relied solely on samples from Western cultures. In the current study, responses from a cross-cultural survey of 30,470 people across 53 nations spanning 11 world regions (North America, Central/South America, Northern Europe, Western Europe, Eastern Europe, Southern Europe, Middle East, Africa, Oceania, Southeast Asia, and East Asia) were used to evaluate whether narcissism (as measured by the Narcissistic Personality Inventory; NPI) was universally associated with short-term mating. Results revealed narcissism scores (including two broad factors and seven traditional facets as measured by the NPI) were functionally equivalent across cultures, reliably associating with key sexual outcomes (e.g., more active pursuit of short-term mating, intimate partner violence, and sexual aggression) and sex-related personality traits (e.g., higher extraversion and openness to experience). Whereas some features of personality (e.g., subjective well-being) were universally associated with socially adaptive facets of Narcissism (e.g., self-sufficiency), most indicators of short-term mating (e.g., unrestricted sociosexuality and marital infidelity) were universally associated with the socially maladaptive facets of narcissism (e.g., exploitativeness). Discussion addresses limitations of these cross-culturally universal findings and presents suggestions for future research into revealing the precise psychological features of narcissism that facilitate the strategic pursuit of short-term mating