bluuebunny/crossref_metadata_2025 huggingface dataset:
Is deagrarianisation real? A study of livelihood activities in rural northern Ghana
Authors: Yaro Joseph Awetori
Published: February 2006

This article examines the livelihoods, portfolios and degree of deagrarianisation of the peasantry in three villages in northern Ghana. It argues that deagrarianisation should be seen as a process embedded in social change, bearing in mind the reversibility between farm and non-farm livelihood strategies used by households (reagrarianisation?). A livelihoods research approach involving qualitative household interviews and quantitative surveys in three villages in the Kassena-Nankani district constitute primary data for this study. Contrary to the deagrarianisation thesis, this study found that livelihood adaptation, implying both a diversification to new or secondary livelihood activities and changing the form, nature and content of the farm sector, characterised rural livelihoods in the area. The adaptation process involves not just a move from the farm to the non-farm sector, but also an intensification of efforts in the farm sector with seasonal diversification into other livelihood activities. The supposedly ‘booming non-farm sector’ is not entirely real, for reasons of marginalisation and exclusion of the poor peasantry, resulting from spatial, capital, infrastructural and market limitations.
Conversation Analysis and language learning
Authors: Seedhouse Paul
Published: February 2006

Interest in Conversation Analysis (CA) and its possible applications in the fields of language learning and language teaching has grown considerably over the last five years. There are now a range of publications which explore this area. The article therefore attempts to synthesise the current state of the research and identify the issues and problems that have arisen and those areas which are suitable for further research. This article starts with a brief introduction to CA methodology and then discusses the range of areas within the broad field of language learning and teaching in which CA has been applied: teaching languages for specific purposes; language teaching materials design; language proficiency assessment; language classroom interaction; NS–NNS (native/non-native speaker) talk; and code-switching. It then discusses the relationship between CA, Applied Linguistics and Second Language Acquisition, and examines the complex issue of what CA can contribute to the study of ‘learning’. The issues are illustrated by an example of a CA analysis of language learning processes. The article proposes that there are now three distinct approaches to the application of CA methodology to the field of language learning and teaching. The article concludes by positioning CA as a social science research methodology and considers possible future directions for research.
On Directing: a Conversation with Katie Mitchell
Authors: Shevtsova Maria
Published: February 2006

One of Britain's foremost directors, Katie Mitchell's career embraces a formidable repertoire of play and opera productions. She has a taste for Greek tragedy – her Phoenician Women (1995) won the Evening Standard Best Director Award – and takes in Gorky, Chekhov, Genet, and Beckett, as well as such contemporaries as Kevin Elyot, whose Forty Winks she directed at the Royal Court in 2004. She has worked in Dublin, Milan, and Stockholm, and is an Associate Director at the National Theatre. This interview with NTQ co-editor Maria Shevtsova shows Mitchell's lucid and passionate engagement with her craft. It took place in London in several stages from December 2004 to July 2005, during a period of intense activity for Mitchell. Maria Shevtsova wishes to thank her for so generously giving her time.
Banditry Unleash'd; or, How The Robbers Reached the Stage
Authors: Brandt George W.
Published: February 2006

Friedrich Schiller – poet, historian, and philosopher as well as dramatist – is acknowledged to be a towering figure in German-language theatre, yet has had only a fitful impact on the stages of the English-speaking world, where such of his works as Don Carlos, Intrigue and Love (Luisa Miller in the operatic version) and William Tell are better known through the filters of Verdi and Rossini than in their original form. But there were signs in 2005 – the bicentenary of Schiller's death at the tragically early age of forty-five – that the English theatre was taking more notice of this major playwright, with Phyllida Lloyd's production of Mary Stuart and Michael Grandage's of Don Carlos both well received. In the article which follows, George W. Brandt traces Schiller's troubled breakthrough into professional theatre as a young man with his first play, The Robbers – which, while significantly different from his later work, does anticipate his lifelong preoccupation with the theme of freedom. George W. Brandt, Senior Research Fellow and Professor Emeritus in the Drama Department of the University of Bristol, has previously contributed to NTQ with articles on Bristol's Shakespeare at the Tobacco Factory Company (NTQ 72), and Iffland's 1796 guest performance in the Weimar of Goethe and Schiller (NTQ 77).
‘The Wooden Heads of the People’: Arnold Daly and Bernard Shaw
Authors: Woods Leigh
Published: February 2006

Once Arnold Daly and Bernard Shaw had got through their baptisms of fire in the transatlantic theatre of the 1890s, the circumstances for their future collaboration must have seemed propitious to them both. However, the Irish-American's inflexibility and the Anglo-Irishman's passion for control led to the fracturing of the relationship within the span of a few years in the first decade of the new century. The exposure of their work – in tandem in American vaudeville and later as competitors on the English variety stage – marked points of their disagreement and quirks in their difficult personalities as they scrambled for audiences who rarely appreciated them as much as both felt they deserved. Leigh Woods, Head of Theatre Studies at the University of Michigan, explores the breakdown of a partnership that launched one man on a course to oblivion and the other on a path to greater glory.
Role for GLUT1 in diabetic glomerulosclerosis
Authors: Heilig Charles W. , Brosius Frank C. , Cunningham Carol
Published: February 2006

Numerous studies have investigated specific pathways that link diabetes and high extracellular glucose exposure to glomerulosclerosis and mesangial cell extracellular matrix production. However, only in the past ten years has a role for glucose transporters in this process been addressed. Many different glucose transporters are expressed in glomeruli; of these, the GLUT1 facilitative glucose transporter is upregulated in the diabetic renal cortex and in response to glomerular hypertension, as well as in cultured mesangial cells exposed to high glucose. Transgenic mouse and cell models have recently been developed to test the role of GLUT1 in the pathogenesis of glomerulosclerosis with and without diabetes. Clinical studies of GLUT1 alleles performed in humans have identified GLUT1 susceptibility alleles for diabetic nephropathy. Studies are also currently under way to assess the potential role of GLUT1 in nondiabetic renal disorders.
Confirmation of Arabia plate slow motion by new GPS data in Yemen
Authors: Vigny Christophe , Huchon Philippe , Ruegg Jean‐Claude , Khanbari Khaled , Asfaw Laike M.
Published: February 2006

During the last 10 years, a network of about 30 GPS sites was measured in Djibouti, East Africa. Additional points were also measured in Yemen, Oman, Ethiopia, Iran, and on La Réunion island. Merged with data from the available International GPS Service permanent stations scattered on the different plates in the area (Eurasia, Anatolia, Africa, Arabia, Somalia), this unique data set provides new insight on the current deformation in the Africa‐Somalia‐Arabia triple junction area and on the Arabian plate motion. Here we show that coherent motions of points in Yemen, Bahrain, Oman, and Iran allow us to estimate a geodetically constrained angular velocity for the Arabian plate (52.59°N, 15.74°W, 0.461°/Myr in ITRF2000). This result differs significantly from earlier determinations and is based upon our vectors in Yemen. They provide new additional data and better geometry for angular velocity determination. Combined with the African and Somalian motions, this new angular velocity results in predicted spreading rates in the Red Sea and the Gulf of Aden which are 15–20% lower than those measured from oceanic magnetic anomalies and thus averaged over the last 3 Myr. With respect to Eurasia, the geodetic motion of Arabia is also about 30% slower than predicted by NUVEL‐1A. On the basis of the kinematic results presented here and on other evidence for a similar slower geodetic rate of the Indian plate, we suggest that the whole collision zone between Africa, Arabia, India on one hand and Eurasia on the other hand has slowed down in the last 3 Myr.
Global temperature calibration of the alkenone unsaturation index (UK′37) in surface waters and comparison with surface sediments
Authors: Conte Maureen H. , Sicre Marie‐Alexandrine , Rühlemann Carsten , Weber John C. , Schulte Sonja , Schulz‐Bull Detlef , Blanz Thomas
Published: February 2006

In this paper, we compile the current surface seawater C37 alkenone unsaturation (UK′37) measurements (n = 629, −1 to 30°C temperature range) to derive a global, field‐based calibration of UK′37 with alkenone production temperature. A single nonlinear “global” surface water calibration of UK′37 accurately predicts alkenone production temperatures over the diversity of modern‐day oceanic environments and alkenone‐synthesizing populations (T = −0.957 + 54.293(UK′37) − 52.894(UK′37)2 + 28.321(UK′37)3, r2 = 0.97, n = 567). The mean standard error of estimation is 1.2°C with insignificant bias in estimated production temperature among the different ocean regions sampled. An exception to these trends is regions characterized by strong lateral advection and extreme productivity and temperature gradients (e.g., the Brazil‐Malvinas Confluence). In contrast to the surface water data, the calibration of UK′37 in surface sediments with overlying annual mean sea surface temperature (AnnO) is best fit by a linear model (AnnO = 29.876(UK′37) − 1.334, r2 = 0.97, n = 592). The standard error of estimation (1.1°C) is similar to that of the surface water production calibration, but a higher degree of bias is observed among the regional data sets. The sediment calibration differs significantly from the surface water calibration. UK′37 in surface sediments is consistently higher than that predicted from AnnO and the surface water production temperature calibration, and the magnitude of the offset increases as the surface water AnnO decreases. We apply the global production temperature calibration to the coretop UK′37 data to estimate the coretop alkenone integrated production temperature (coretop IPT) and compare this with the overlying annual mean sea surface temperature (AnnO). We use simple models to explore the possible causes of the deviation observed between the coretop temperature signal, as estimated by UK′37, and AnnO. Our results indicate that the deviation can best be explained if seasonality in production and/or thermocline production as well as differential degradation of 37:3 and 37:2 alkenones both affect the sedimentary alkenone signal.
Rupture length and duration of the 2004 Aceh‐Sumatra earthquake from the phases of the Earth's gravest free oscillations
Authors: Lambotte S. , Rivera L. , Hinderer J.
Published: February 2006

The Aceh‐Sumatra 2004 earthquake strongly excited the Earth's free oscillations. Well separated split multiplets provide useful information on the earthquake source. Particularly, the phases of split singlets constrain the duration, rupture length and mean rupture velocity. We analyze the initial phases of some of the Earth's gravest free oscillations (0S2, 0S3, 0S0 and 1S0) in order to constrain the space‐time finiteness of the source. We use recordings of vertical broadband seismometers and superconducting gravimeters from several worldwide geophysical networks. We estimate a rupture length of about 1220 km, a source time duration of about 500 s, and a mean rupture velocity of 2.4 km/s.
Shear‐wave splitting observations across southwest Ireland
Authors: Do V. C. , Readman P. W. , O'Reilly B. M. , Landes M.
Published: February 2006

Shear‐wave splitting analyses have been carried out on teleseismic data from the southwest of Ireland acquired during the Irish Seismological Lithospheric Experiment (ISLE). The data were gathered over a ten‐month period by a temporary network of 23 broadband and short‐period stations. The results are compared with data from two permanent broadband seismic stations, which have recorded SKS and SKKS phases for up to 10 years. The purpose of the experiment was to investigate possible anisotropy within the crust and mantle related to Caledonian deformation. Here we report splitting results which show an average delay time of 1.2 s and a variation of fast‐polarisation direction with back azimuth that surprisingly suggests a much deeper origin for anisotropy than was anticipated.
Cloud‐to‐ground lightning downwind of the 2002 Hayman forest fire in Colorado
Authors: Lang Timothy J. , Rutledge Steven A.
Published: February 2006

The Hayman forest fire occurred near Denver, burning ∼138,000 acres during 8 June–2 July 2002. It produced aerosol that filled the troposphere over Colorado, allowing an investigation of the effects of increased aerosol concentration on cloud‐to‐ground (CG) lightning polarity. Compared to climatological values over eastern Colorado, the 2002 fire period showed enhancements in satellite‐derived aerosol optical depth (AOD; up to 0.4 units greater), and the percentage of +CG lightning more than doubled in the domain average. However, spatial patterns were different in the fire's vicinity, with increases in AOD but not in +CGs. Denver soundings during the fire were compared to climatology, and showed environmental differences that are associated with increased +CG percentage, in particular higher cloud base and reduced warm‐cloud depth. Thus, our findings provide only mixed support for the idea that smoke aerosols impact CG polarity, and suggest a possible link between drought conditions and increased +CG percentage.
Influence of stratosphere‐to‐troposphere exchange on the seasonal cycle of surface ozone at Mount Waliguan in western China
Authors: Ding Aijun , Wang Tao
Published: February 2006

Surface ozone over the Qinghai‐Tibetan Plateau exhibits a summertime maximum contrasting to a spring peak observed at many remote locations in the Northern Hemisphere. In this study we conducted meteorological simulations for April–August 2003, when intensive measurement of trace gases was carried out at remote Mount Waliguan, to elucidate the influence of atmospheric dynamics on the observed ozone events and seasonal cycle. Examinations of potential vorticity and trace gases suggest that most of the synoptic‐scale ozone enhancements were due to stratospheric intrusions, as opposed to transport of anthropogenic pollutions. Case studies show that the existence of jet stream is the main dynamical cause for these intrusions. Further analysis of upper‐level mean zonal winds suggests that stronger subtropical jet streams lead to more frequent stratospheric intrusions in summer compared to spring, which may have contributed to the higher summertime surface ozone there and possibly also in other regions of Central Asia.
El Niño–Southern Oscillation influences on the interannual variability of leaf appearance dates in central Siberia
Authors: Vicente‐Serrano Sergio M. , Delbart Nicolas , Le Toan Thuy , Grippa Manuela
Published: February 2006

This paper analyses the role of the El Niño–Southern Oscillation (ENSO) on the interannual variability of the leaf appearance dates of boreal forests in the Northern Hemisphere (1982–2004). We find significant negative correlations between the ENSO, quantified by means of the Southern Oscillation Index (SOI), and the leaf appearance dates in central Siberia with up to 9 months lag. The correlations between leaf appearance dates and summer Sea Surface Temperatures (SST) show a pattern that resembles the ENSO phenomena with positive and significant correlations in the East Pacific and negative in the West. These findings are explained by the role of SOI on Sea Level Pressures, 500 hPa Geopotential and the wind flow direction and intensity during the months of leaf appearance (April and May) and on average temperatures, which determine noticeably the date of leaf appearance.
Evidence for floatation or near floatation in the mouth of Kamb Ice Stream, West Antarctica, prior to stagnation
Authors: Catania G. A. , Conway H. , Raymond C. F. , Scambos T. A.
Published: February 2006

Ice‐penetrating radar profiles reveal distinctive internal structure within the flat ice terrains that bound the lower reaches of the now stagnant Kamb Ice Stream, West Antarctica; slightly warped but continuous upper layers overlie extensive deep line diffractors located at a uniform depth several hundreds of meters above the bed. On the basis of their orientation and morphology we interpret the line diffractors to be the tips of basal crevasses. At the boundary between these terrains and the inter–ice stream ridges, internal layers are strongly downwarped, and the deepest layers are truncated at the bed. Results from simple kinematic ice flow modeling indicate that the observed layer pattern requires a cumulative basal melt of 120–350 m over a time period that is less than 300 years. Fields of basal crevasses are typically observed in ice shelves; however, isolated basal crevasses can also be found in grounded ice when the subglacial water pressure is close to overburden. On the basis of the presence of these crevasses we argue that these terrains were once floating or near floating in the past. We combine model results with estimates of recent ice thickness change to suggest that floatation was transitory and the region may have become fully grounded roughly 200 years prior to stagnation of Kamb Ice Stream.
Ramifications of increased salinity in tidal freshwater sediments: Geochemistry and microbial pathways of organic matter mineralization
Authors: Weston Nathaniel B. , Dixon Ray E. , Joye Samantha B.
Published: February 2006

The effects of salinity intrusion on the anaerobic microbial and geochemical dynamics of tidal freshwater sediments were investigated using flow‐through sediment reactors. In freshwater control sediments, organic matter mineralization was dominated by methanogenesis (62%), followed by sulfate reduction (18%), denitrification (10%), and iron reduction (10%). Upon salinity intrusion, nutrient (ammonium, silicate, phosphate) concentrations increased and rates of methanogenesis declined. Iron‐oxide bioavailability increased and microbial iron reduction appeared to account for >60% of organic matter oxidation for several days after salinity intrusion. However, sulfate reduction was the dominant pathway (>50%) of organic matter oxidation within 2 weeks of salinity intrusion, and accounted for >95% of total organic matter mineralization after 4 weeks. Total in situ sediment organic matter mineralization doubled following salinity intrusion. Increased nutrient release, decreased methanogenesis and a rapid shift to sulfate reduction, with a coincident increase overall organic matter mineralization, accompanied salinity intrusion into previously freshwater riverine sediments.
How northern peatlands influence the Earth's radiative budget: Sustained methane emission versus sustained carbon sequestration
Authors: Frolking Steve , Roulet Nigel , Fuglestvedt Jan
Published: February 2006

Northern peatlands sequester carbon and emit methane, and thus have both cooling and warming impacts on the climate system through their influence on atmospheric burdens of CO2 and CH4. These competing impacts are usually compared by the global warming potential (GWP) methodology, which determines the equivalent CO2 annual emission that would have the same integrated radiative forcing impact over a chosen time horizon as the annual CH4 emission. We use a simple model of CH4 and CO2 pools in the atmosphere to extend this analysis to quantify the dynamics, over years to millennia, of the net radiative forcing impact of a peatland that continuously emits CH4 and sequesters C. We find that for observed ratios of CH4 emission to C sequestration (roughly 0.1–2 mol mol−1), the radiative forcing impact of a northern peatland begins, at peatland formation, as a net warming that peaks after about 50 years, remains a diminishing net warming for the next several hundred to several thousand years, depending on the rate of C sequestration, and thereafter is or will be an ever increasing net cooling impact. We then use the model to evaluate the radiative forcing impact of various changes in CH4 and/or CO2 emissions. In all cases, the impact of a change in CH4 emissions dominates the radiative forcing impact in the first few decades, and then the impact of the change in CO2 emissions slowly exerts its influence.
Mutations in dopachrome tautomerase (Dct) affect eumelanin/pheomelanin synthesis, but do not affect intracellular trafficking of the mutant protein
Authors: Costin Gertrude-E. , Valencia Julio C. , Wakamatsu Kazumasa , Ito Shosuke , Solano Francisco , Milac Adina L. , Vieira Wilfred D. , Yamaguchi Yuji , Rouzaud François , Petrescu Andrei-J. , Lamoreux M. Lynn , Hearing Vincent J.
Published: February 2006

Dopachrome tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and in the rapid metabolism of its toxic intermediates. Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn congenic C57BL/6J non-agouti black mice and were used to study the effect(s) of these mutations on the intracellular trafficking of Dct and on the pigmentation of the cells. Dct activity is 3-fold lower in slaty cells compared with non-agouti black melanocytes, whereas slaty light melanocytes have a surprisingly 28-fold lower Dct activity. Homology modelling of the active site of Dct suggests that the slaty mutation [R194Q (Arg194→Gln)] is located in the active site and may alter the ability of the enzyme to transform the substrate. Transmembrane prediction methods indicate that the slaty light mutation [G486R (Gly486→Arg)] may result in the sliding of the transmembrane domain towards the N-terminus, thus interfering with Dct function. Chemical analysis showed that both Dct mutations increase pheomelanin and reduce eumelanin produced by melanocytes in culture. Thus the enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis.
The GTPase RhoA increases utrophin expression and stability, as well as its localization at the plasma membrane
Authors: Bonet-Kerrache Armelle , Fortier Mathieu , Comunale Franck , Gauthier-Rouvière Cécile
Published: February 2006

The Rho family of small GTPases are signalling molecules involved in cytoskeleton remodelling and gene transcription. Their activities are important for many cellular processes, including myogenesis. In particular, RhoA positively regulates skeletal-muscle differentiation. We report in the present study that the active form of RhoA increases the expression of utrophin, the autosomal homologue of dystrophin in the mouse C2C12 and rat L8 myoblastic cell lines. Even though this RhoA-dependent utrophin increase is higher in proliferating myoblasts, it is maintained during myogenic differentiation. This occurs via two mechanisms: (i) transcriptional activation of the utrophin promoter A and (ii) post-translational stabilization of utrophin. In addition, RhoA increases plasma-membrane localization of utrophin. Thus RhoA activation up-regulates utrophin levels and enhances its localization at the plasma membrane.
Effects of nitric oxide donors on cybrids harbouring the mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) A3243G mitochondrial DNA mutation
Authors: Sandhu Jagdeep K. , Sodja Caroline , Mcrae Kevan , Li Yan , Rippstein Peter , Wei Yau-Huei , Lach Boleslaw , Lee Fay , Bucurescu Septimiu , Harper Mary-Ellen , Sikorska Marianna
Published: February 2006

Reactive nitrogen and oxygen species (O2•−, H2O2, NO• and ONOO−) have been strongly implicated in the pathophysiology of neurodegenerative and mitochondrial diseases. In the present study, we examined the effects of nitrosative and/or nitrative stress generated by DETA-NO {(Z)-1-[2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate}, SIN-1 (3-morpholinosydnonimine hydrochloride) and SNP (sodium nitroprusside) on U87MG glioblastoma cybrids carrying wt (wild-type) and mutant [A3243G (Ala3243→Gly)] mtDNA (mitochondrial genome) from a patient suffering from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). The mutant cybrids had reduced activity of cytochrome c oxidase, significantly lower ATP level and decreased mitochondrial membrane potential. However, endogenous levels of reactive oxygen species were very similar in all cybrids regardless of whether they carried the mtDNA defects or not. Furthermore, the cybrids were insensitive to the nitrosative and/or nitrative stress produced by either DETA-NO or SIN-1 alone. Cytotoxicity, however, was observed in response to SNP treatment and a combination of SIN-1 and glucose-deprivation. The mutant cybrids were significantly more sensitive to these insults compared with the wt controls. Ultrastructural examination of dying cells revealed several characteristic features of autophagic cell death. We concluded that nitrosative and/or nitrative stress alone were insufficient to trigger cytotoxicity in these cells, but cell death was observed with a combination of metabolic and nitrative stress. The vulnerability of the cybrids to these types of injury correlated with the cellular energy status, which were compromised by the MELAS mutation.
Regulation of FAK Ser-722 phosphorylation and kinase activity by GSK3 and PP1 during cell spreading and migration
Authors: Bianchi Mariarita , De Lucchini Stefania , Marin Oriano , Turner David L. , Hanks Steven K. , Villa-Moruzzi Emma
Published: February 2006

In addition to tyrosine sites, FAK (focal adhesion kinase) is phosphorylated on multiple serine residues. In the present study, the regulation of two of these sites, Ser-722 (S1) and Ser-911 (S4), was investigated. Phosphorylation of S1 (but not S4) decreased in resuspended cells, and recovered during spreading on fibronectin, indicating adhesion-dependent regulation. GSK3 (glycogen synthase kinase 3) inhibitors decreased S1 phosphorylation, and siRNA (short interfering RNA) silencing indicated further the involvement of GSK3β. Furthermore, GSK3β was found to become activated during cell spreading on fibronectin, and to physically associate with FAK. S1 phosphorylation was observed to decrease in wounded cell monolayers, while GSK3β underwent inactivation and later was observed to increase to the original level within 24 h. Direct phosphorylation of S1, requiring pre-phosphorylation of Ser-726 in the +4 position, was demonstrated using purified GSK3 and a synthetic peptide containing FAK residues 714–730. An inhibitory role for S1 phosphorylation in FAK signalling was indicated by findings that both alanine substitution for S1 and dephosphorylation of S1 by PP1 (serine/threonine protein phosphatase type-1) resulted in an increase in FAK kinase activity; likewise, this role was also shown by cell treatment with the GSK3 inhibitor LiCl. The inhibitory role was confirmed by the finding that cells expressing FAK with alanine substitution for S1 displayed improved cell spreading and faster migration in wound-healing and trans-well assays. Finally, the finding that S1 phosphorylation increased in cells treated with the PP1 inhibitor okadaic acid indicated targeting of this site by PP1. These results indicate an additional mechanism for regulation of FAK activity during cell spreading and migration, involving Ser-722 phosphorylation modulated through the competing actions of GSK3β and PP1.
A novel α-glucosidase from the acidophilic archaeonFerroplasma acidiphilumstrain Y with high transglycosylation activity and an unusual catalytic nucleophile
Authors: Ferrer Manuel , Golyshina Olga V. , Plou Francisco J. , Timmis Kenneth N. , Golyshin Peter N.
Published: February 2006

Ferroplasma acidiphilum strain Y (DSM 12658), a ferrous iron-oxidizing, acidophilic and mesophilic archaeon, was found to produce a membrane-bound α-glucosidase (αGluFa) showing no significant similarity to any of the known glycoside hydrolases classified in different families and having an unusual catalytic site consisting of a threonine and a histidine residue. The highest α-glucosidase activity was found at low pH, 2.4–3.5, and the substrate preference order was: sucrose>maltose>maltotriose ≫maltotetraose≫malto-oligosaccharides from maltopentaose to maltoheptaose⋙soluble starch (kcat/Km was 293.0, 197.0, 18.8, 0.3 and 0.02 s−1·mM−1 respectively). The enzyme was able to transfer glucosyl groups from maltose as donor, to produce exclusively maltotriose (up to 300 g/l). Chemical modification and electrospray ionization MS analysis of 5-fluoro-α-D-glucopyranosyl-enzyme derivatives, coupled with site-directed mutagenesis, strongly suggested that the putative catalytic nucleophile in this enzyme is Thr212. Iron was found to be essential for enzyme activity and integrity, and His390 was shown to be essential for iron binding. These results suggest that the metalloenzyme αGluFa is a new member of the glycosyl hydrolase family that uses a novel mechanism for sugar glycosylation and/or transglycosylation.
Structural and functional differences in isoforms of mouse major urinary proteins: a male-specific protein that preferentially binds a male pheromone
Authors: Armstrong Stuart D. , Robertson Duncan H. L. , Cheetham Sarah A. , Hurst Jane L. , Beynon Robert J.
Published: February 2006

The MUPs (major urinary proteins) of the house mouse, Mus domesticus, are lipocalins that bind and slowly release male-specific pheromones in deposited scent marks. However, females also express these proteins, consistent with a second role in encoding individual signatures in scent marks. We have purified and characterized an atypical MUP from the urine of male C57BL/6J inbred mice, which is responsible for the binding of most of the male pheromone, 2-sec-butyl-4,5-dihydrothiazole, and which is also responsible for the slow release of this pheromone from scent marks. This protein is absent from the urine of female mice of the same strain. The protein has been characterized by MS, leading to unequivocal identification as a previously uncharacterized gene product, providing compelling evidence for the expression of this gene in liver and manifestation in urine. These properties contrast strongly with those of the other MUPs in the same urine sample, and suggest that the requirement to manifest a male-specific pheromone has been met by evolution of a cognate protein specifically adapted to the binding and release of this ligand. This atypical MUP is also present in a random sample of wild-caught male mice, confirming that this protein is not specific to the inbred mouse strain but is present in natural populations also.
Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeastSaccharomyces cerevisiae
Authors: Petrovič Uroš , Šribar Jernej , Matis Maja , Anderluh Gregor , Peter-Katalinić Jasna , Križaj Igor , Gubenšek Franc
Published: February 2006

Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to γ and ε isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.
Investigating the allosterism of acyl-CoA:cholesterol acyltransferase (ACAT) by using various sterols: in vitro and intact cell studies
Authors: Liu Jay , Chang Catherine C. Y. , Westover Emily J. , Covey Douglas F. , Chang Ta-Yuan
Published: February 2006

ACAT1 (acyl-CoA:cholesterol acyltransferase 1) is thought to have two distinct sterol-binding sites: a substrate-binding site and an allosteric-activator site. In the present work, we investigated the structural features of various sterols as substrates and/or activators in vitro. The results show that without cholesterol, the plant sterol sitosterol is a poor substrate for ACAT. In the presence of cholesterol, ACAT1-mediated esterification of sitosterol is highly activated while ACAT2-mediated esterification of sitosterol is only moderately activated. For ACAT1, we show that the stereochemistry of the 3-hydroxy group at steroid ring A is a critical structural feature for a sterol to serve as a substrate, but less critical for activation. Additionally, enantiomeric cholesterol, which has the same biophysical properties as cholesterol in membranes, fails to activate ACAT1. Thus ACAT1 activation by cholesterol is the result of stereo-specific interactions between cholesterol and ACAT1, and is not related to the biophysical properties of phospholipid membranes. To demonstrate the relevance of the ACAT1 allosteric model in intact cells, we showed that sitosterol esterification in human macrophages is activated upon cholesterol loading. We further show that the activation is not due to an increase in ACAT1 protein content, but is partly due to an increase in the cholesterol content in the endoplasmic reticulum where ACAT1 resides. Together, our results support the existence of a distinct sterol-activator site in addition to the sterol-substrate site of ACAT1 and demonstrate the applicability of the ACAT1 allosteric model in intact cells.
H-NS is a part of a thermally controlled mechanism for bacterial gene regulation
Authors: Ono Shusuke , Goldberg Martin D. , Olsson Tjelvar , Esposito Diego , Hinton Jay C. D. , Ladbury John E.
Published: February 2006

Temperature is a primary environmental stress to which micro-organisms must be able to adapt and respond rapidly. Whereas some bacteria are restricted to specific niches and have limited abilities to survive changes in their environment, others, such as members of the Enterobacteriaceae, can withstand wide fluctuations in temperature. In addition to regulating cellular physiology, pathogenic bacteria use temperature as a cue for activating virulence gene expression. This work confirms that the nucleoid-associated protein H-NS (histone-like nucleoid structuring protein) is an essential component in thermoregulation of Salmonella. On increasing the temperature from 25 to 37 °C, more than 200 genes from Salmonella enterica serovar Typhimurium showed H-NS-dependent up-regulation. The thermal activation of gene expression is extremely rapid and change in temperature affects the DNA-binding properties of H-NS. The reduction in gene repression brought about by the increase in temperature is concomitant with a conformational change in the protein, resulting in the decrease in size of high-order oligomers and the appearance of increasing concentrations of discrete dimers of H-NS. The present study addresses one of the key complex mechanisms by which H-NS regulates gene expression.
Zn2+-induced reversible dissociation of subunit Rpn10/p54 of theDrosophila26 S proteasome
Authors: Kiss Petra , Szabó Áron , Hunyadi-Gulyás Éva , Medzihradszky Katalin F. , Lipinszki Zoltán , Pál Margit , Udvardy Andor
Published: February 2006

In the presence of Zn2+, the Drosophila 26 S proteasome disassembles into RP (regulatory particle) and CP (catalytic particle), this process being accompanied by the dissociation of subunit Rpn10/p54, the ubiquitin receptor subunit of the proteasome. The dissociation of Rpn10/p54 induces extensive rearrangements within the lid subcomplex of the RP, while the structure of the ATPase ring of the base subcomplex seems to be maintained. As a consequence of the dissociation of the RP, the peptidase activity of the 26 S proteasome is lost. The Zn2+-induced structural and functional changes are fully reversible; removal of Zn2+ is followed by reassociation of subunit Rpn10/p54 to the RP, reassembly of the 26 S proteasome and resumption of the peptidase activity. After the Zn2+-induced dissociation, Rpn10/p54 interacts with a set of non-proteasomal proteins. Hsp82 (heat-shock protein 82) has been identified by MS as the main Rpn10/p54-interacting protein, suggesting its role in the reassembly of the 26 S proteasome after Zn2+ removal. The physiological relevance of another Rpn10/p54-interacting protein, the Smt3 SUMO (small ubiquitin-related modifier-1)-activating enzyme, detected by chemical cross-linking, has been confirmed by yeast two-hybrid analysis. Besides the Smt3 SUMO-activating enzyme, the Ubc9 SUMO-conjugating enzyme also exhibited in vivo interaction with the 5′-half of Rpn10/p54 in yeast cells. The mechanism of 26 S proteasome disassembly after ATP depletion is clearly different from that induced by Zn2+. Rpn10/p54 is permanently RP-bound during the ATP-dependent assembly–disassembly cycle, but during the Zn2+ cycle it reversibly shuttles between the RP-bound and free states.
Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity
Authors: Raikwar Nandita S. , Bowen Rosario F. , Deeg Mark A.
Published: February 2006

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal β-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn2+, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity.
Slx4 becomes phosphorylated after DNA damage in a Mec1/Tel1-dependent manner and is required for repair of DNA alkylation damage
Authors: Flott Sonja , Rouse John
Published: February 2006

Members of the RecQ family of DNA helicases, mutated in several syndromes associated with cancer predisposition, are key regulators of genome stability. The Saccharomyces cerevisiae SLX4 gene is required for cell viability in the absence of Sgs1, the only yeast RecQ helicase. SLX4 encodes one subunit of the heterodimeric Slx1–Slx4 endonuclease, although its cellular function is not clear. Slx1–Slx4 was reported to preferentially cleave replication fork-like structures in vitro, and cells lacking SLX4 are hypersensitive to DNA alkylation damage. Here we report that Slx4 becomes phosphorylated in cells exposed to a wide range of genotoxins. Even though it has been proposed that the role of Slx4 is restricted to S-phase, Slx4 phosphorylation is observed in cells arrested in G1 or G2 phases of the cell cycle, but not during an unperturbed cell cycle. Slx4 phosphorylation is completely abolished in cells lacking the Mec1 and Tel1 protein kinases, critical regulators of genome stability, but is barely affected in the absence of both Rad53 and Chk1 kinases. Finally we show that, whereas both Slx1 and Slx4 are dispensable for activation of cell-cycle checkpoints, Slx4, but not Slx1, is required for repair of DNA alkylation damage in both aynchronously growing cells and in G2-phase-arrested cells. These results reveal Slx4 as a new target of the Mec1/Tel1 kinases, with a crucial role in DNA repair that is not restricted to the processing of stalled replisomes.
A direct, continuous, sensitive assay for protein disulphide-isomerase based on fluorescence self-quenching
Authors: Raturi Arun , Vacratsis Panayiotis O. , Seslija Dana , Lee Lana , Mutus Bulent
Published: February 2006

PDI (protein disulphide-isomerase) activity is generally monitored by insulin turbidity assay or scrambled RNase assay, both of which are performed by UV–visible spectroscopy. In this paper, we present a sensitive fluorimetric assay for continuous determination of disulphide reduction activity of PDI. This assay utilizes the pseudo-substrate diabz-GSSG [where diabz stands for di-(o-aminobenzoyl)], which is formed by the reaction of isatoic anhydride with the two free N-terminal amino groups of GSSG. The proximity of two benzoyl groups leads to quenching of the diabz-GSSG fluorescence by approx. 50% in comparison with its non-disulphide-linked form, abz-GSH (where abz stands for o-aminobenzoyl). Therefore the PDI-dependent disulphide reduction can be monitored by the increase in fluorescence accompanying the loss of proximity-quenching upon conversion of diabz-GSSG into abz-GSH. The apparent Km of PDI for diabz-GSSG was estimated to be approx. 15 μM. Unlike the insulin turbidity assay and scrambled RNase assay, the diabz-GSSG-based assay was shown to be effective in determining a single turnover of enzyme in the absence of reducing agents with no appreciable blank rates. The assay is simple to perform and very sensitive, with an estimated detection limit of approx. 2.5 nM PDI, enabling its use for the determination of platelet surface PDI activity in crude sample preparations.
Tumour necrosis factor α induces co-ordinated activation of rat GSH synthetic enzymes via nuclear factor κB and activator protein-1
Authors: Yang Heping , Magilnick Nathaniel , Ou Xiaopeng , Lu Shelly C.
Published: February 2006

GSH synthesis occurs via two enzymatic steps catalysed by GCL [glutamate–cysteine ligase, made up of GCLC (GCL catalytic subunit), and GCLM (GCL modifier subunit)] and GSS (GSH synthetase). Co-ordinated up-regulation of GCL and GSS further enhances GSH synthetic capacity. The present study examined whether TNFα (tumour necrosis factor α) influences the expression of rat GSH synthetic enzymes. To facilitate transcriptional studies of the rat GCLM, we cloned its 1.8 kb 5′-flanking region. TNFα induces the expression and recombinant promoter activities of GCLC, GCLM and GSS in H4IIE cells. TNFα induces NF-κB (nuclear factor κB) and AP-1 (activator protein 1) nuclear-binding activities. Blocking AP-1 with dominant negative c-Jun or NF-κB with IκBSR (IκB super-repressor, where IκB stands for inhibitory κB) lowered basal expression and inhibited the TNFα-mediated increase in mRNA levels of all three genes. While all three genes have multiple AP-1-binding sites, only GCLC has a NF-κB-binding site. Overexpression with p50 or p65 increased c-Jun mRNA levels, c-Jun-dependent promoter activity and the promoter activity of GCLM and GSS. Blocking NF-κB also lowered basal c-Jun expression and blunted the TNFα-mediated increase in c-Jun mRNA levels. TNFα treatment resulted in increased c-Jun and Nrf2 (nuclear factor erythroid 2-related factor 2) nuclear binding to the antioxidant response element of the rat GCLM and if this was prevented, TNFα no longer induced the GCLM promoter activity. In conclusion, both c-Jun and NF-κB are required for basal and TNFα-mediated induction of GSH synthetic enzymes in H4IIE cells. While NF-κB may exert a direct effect on the GCLC promoter, it induces the GCLM and GSS promoters indirectly via c-Jun.
UDP-glucose dehydrogenases of maize: a role in cell wall pentose biosynthesis
Authors: Kärkönen Anna , Murigneux Alain , Martinant Jean-Pierre , Pepey Elodie , Tatout Christophe , Dudley Bernard J. , Fry Stephen C.
Published: February 2006

UDPGDH (UDP-D-glucose dehydrogenase) oxidizes UDP-Glc (UDP-D-glucose) to UDP-GlcA (UDP-D-glucuronate), the precursor of UDP-D-xylose and UDP-L-arabinose, major cell wall polysaccharide precursors. Maize (Zea mays L.) has at least two putative UDPGDH genes (A and B), according to sequence similarity to a soya bean UDPGDH gene. The predicted maize amino acid sequences have 95% similarity to that of soya bean. Maize mutants with a Mu-element insertion in UDPGDH-A or UDPGDH-B were isolated (udpgdh-A1 and udpgdh-B1 respectively) and studied for changes in wall polysaccharide biosynthesis. The udpgdh-A1 and udpgdh-B1 homozygotes showed no visible phenotype but exhibited 90 and 60–70% less UDPGDH activity respectively than wild-types in a radiochemical assay with 30 μM UDP-glucose. Ethanol dehydrogenase (ADH) activity varied independently of UDPGDH activity, supporting the hypothesis that ADH and UDPGDH activities are due to different enzymes in maize. When extracts from wild-types and udpgdh-A1 homozygotes were assayed with increasing concentrations of UDP-Glc, at least two isoforms of UDPGDH were detected, having Km values of approx. 380 and 950 μM for UDP-Glc. Leaf and stem non-cellulosic polysaccharides had lower Ara/Gal and Xyl/Gal ratios in udpgdh-A1 homozygotes than in wild-types, whereas udpgdh-B1 homozygotes exhibited more variability among individual plants, suggesting that UDPGDH-A activity has a more important role than UDPGDH-B in UDP-GlcA synthesis. The fact that mutation of a UDPGDH gene interferes with polysaccharide synthesis suggests a greater importance for the sugar nucleotide oxidation pathway than for the myo-inositol pathway in UDP-GlcA biosynthesis during post-germinative growth of maize.
Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60
Authors: Wadhwa Renu , Takano Syuichi , Kaur Kamaljit , Aida Satoshi , Yaguchi Tomoko , Kaul Zeenia , Hirano Takashi , Taira Kazunari , Kaul Sunil C.
Published: February 2006

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.