bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-native English-speaking English language teachers: History and research
Authors: Moussu Lucie , Llurda Enric
Published: June 2008

Although the majority of English language teachers worldwide are non-native English speakers, no research was conducted on these teachers until recently. After the pioneering work of Robert Phillipson in 1992 and Peter Medgyes in 1994, nearly a decade had to elapse for more research to emerge on the issues relating to non-native English teachers. The publication in 1999 of George Braine's bookNonnative educators in English language teachingappears to have encouraged a number of graduate students and scholars to research this issue, with topics ranging from teachers' perceptions of their own identity to students' views and aspects of teacher education. This article compiles, classifies, and examines research conducted in the last two decades on this topic, placing a special emphasis on World Englishes concerns, methods of investigation, and areas in need of further attention.
Making connections through texts in language teaching
Authors: Kern Richard
Published: June 2008

Language is not just a tool for communication. It is also a resource for creative thought, a framework for understanding the world, a key to new knowledge and human history, and a source of pleasure and inspiration. The Connections Standard is about linking language and literature study to other disciplines (for example, art, music, film, history, among others) and about getting students to experience unique viewpoints available only through a particular language and its cultures. This presentation will argue for the importance of analyzing texts (written, oral, visual, audio-visual) in language teaching. The goal is to give students the chance to position themselves in relation to distinct viewpoints and distinct cultures and to make connections between language and other symbolic ways of making meaning, connections between language and other disciplines, and connections between language and culture. These connections are not easy to make, but they are essential if we are to prepare our students for the broadest range of language use and allow them to achieve their full communicative potential.
The European Centre for Modern Languages: Recent projects
Authors: Slivensky Susanna
Published: June 2008

The hallmark of the Council of Europe's European Centre for Modern Languages (ECML) is the organisation of international language education projects within four-year programmes. Coordinated by European teams, these projects primarily target language experts involved in national and international developments, in associations and in institutions working to enhance standards in language education.
The ‘open-earedness’ hypothesis and the development of age-related aesthetic reactions to music in elementary school children
Authors: Kopiez Reinhard , Lehmann Marco
Published: June 2008

This study investigates age-related changes in musical preference in elementary school children. The tolerance towards unconventional musical styles has been called ‘open-earedness’ (Hargreaves, 1982a), and it is assumed to decline with increasing age. Musical preferences of 186 students from grade 1 to 4 (age range: 6–10 years) were measured by using a 5-point iconographic rating scale. Eight sound examples were presented in a sound questionnaire. Results showed a decline of open-earedness for unconventional music (classical, ethnic and avant-garde music) from grade 1 to 2 (age: 7–8 years). However, this effect disappeared when classical music was excluded from data analysis. Only a few absolute rejections of unconventional musical styles were found, and the mean preference ratings did not exceed the neutral mean range. Future studies will have to consider additional factors of influence to make clear predictions about the point in time when this transition occurs.
Exploring the content of instrumental lessons and gender relations in Australian higher education
Authors: Zhukov Katie
Published: June 2008

This observational study analysed the lesson content of 24 instrumental lessons (piano, strings and winds) using a gender-balanced sample (equal numbers of male/female teachers and students) from five Australian higher education institutions to ascertain the priorities of topics in advanced applied music lessons in the Western Classical tradition. The results were analysed according to gender to determine differences of approach between male and female teachers and male and female students. Same-gender and different-gender pairings were also considered. Technique was found to be of the greatest importance, followed by Articulation and Expression. Some gender differences have emerged between the teachers, with the male teachers tending towards a more analytical approach and the female teachers adopting more balanced lesson content. The treatment of students showed some divergence, with greater emphasis on Expression in the lessons of female students, whereas the male students studied more Structure. The results demonstrate stereotypical gender behaviour among the teachers and towards their students not previously observed in this educational setting.
Sharing the podium: exploring the process of peer learning in professional conducting
Authors: Bartleet Brydie-Leigh , Hultgren Ralph
Published: June 2008

We discuss a recent peer-learning project we undertook as co-conductors of the Young Conservatorium Wind Orchestra at Griffith University. Drawing on current educational theory on peer learning and material from our conducting practice and research, we explore how this approach offers professional conductors the opportunity to work together in an inclusive and empowering learning environment. We outline our peer learning context, the learning relationship we shared, the most significant musical outcomes of such a process, and the implications for conducting pedagogy and the professional development of conductors.
ESTIMATING PANEL DATA DURATION MODELS WITH CENSORED DATA
Authors: Lee Sokbae
Published: June 2008

This paper presents a method for estimating a class of panel data duration models, under which an unknown transformation of the duration variable is linearly related to the observed explanatory variables and the unobserved heterogeneity (or frailty) with completely known error distributions. This class of duration models includes a panel data proportional hazards model with fixed effects. The proposed estimator is shown to ben1/2-consistent and asymptotically normal with dependent right censoring. The paper provides some discussions on extending the estimator to the cases of longer panels and multiple states. Some Monte Carlo studies are carried out to illustrate the finite-sample performance of the new estimator.
ERGODICITY, MIXING, AND EXISTENCE OF MOMENTS OF A CLASS OF MARKOV MODELS WITH APPLICATIONS TO GARCH AND ACD MODELS
Authors: Meitz Mika , Saikkonen Pentti
Published: June 2008

This paper studies a class of Markov models that consist of two components. Typically, one of the components is observable and the other is unobservable or “hidden.” Conditions under which geometric ergodicity of the unobservable component is inherited by the joint process formed of the two components are given. This implies existence of initial values such that the joint process is strictly stationary and β-mixing. In addition to this, conditions for the existence of moments are also obtained, and extensions to the case of nonstationary initial values are provided. All these results are applied to a general model that includes as special cases various first-order generalized autoregressive conditional heteroskedasticity (GARCH) and autoregressive conditional duration (ACD) models with possibly complicated nonlinear structures. The results only require mild moment assumptions and in some cases provide necessary and sufficient conditions for geometric ergodicity.
A devil's staircase from rotations and irrationality measures for Liouville numbers
Authors: KWON DOYONG
Published: June 2008

From Sturmian and Christoffel words we derive a strictly increasing function Δ:[0,∞) → . This function is continuous at every irrational point, while at rational points, left-continuous but not right-continuous. Moreover, it assumes algebraic integers at rationals, and transcendental numbers at irrationals. We also see that the differentiation of Δ distinguishes some irrationality measures of real numbers.
The processing of German word stress: evidence for the prosodic hierarchy
Authors: Domahs Ulrike , Wiese Richard , Bornkessel-Schlesewsky Ina , Schlesewsky Matthias
Published: June 2008

The present paper explores whether the metrical foot is necessary for the description of prosodic systems. To this end, we present empirical findings on the perception of German word stress using event-related brain potentials as the dependent measure. A manipulation of the main stress position within three-syllable words revealed differential brain responses, which (a) correlated with the reorganisation of syllables into feet in stress violations, and (b) differed in strength depending on syllable weight. The experiments therefore provide evidence that the processing of word stress not only involves lexical information about stress positions, but also (quantity-sensitive) information about metrical structures, in particular feet and syllables.
Generative phonology in the late 1940s
Authors: Goldsmith John A.
Published: June 2008

This paper offers a careful reading of an article published by Rulon Wells in Language in 1949 on the subject of automatic alternations in phonology. Read with a modern eye, it reveals that phonologists were exploring the value and use of phonological derivations, including both abstract representations and intermediate representations, in the late 1940s. Contrary to what has been suggested in the literature, Bloomfield's explorations in rule ordering published in 1939 were not isolated and without influence. Our conclusion is the null hypothesis: that there is an intellectual continuity from the work of Sapir and Bloomfield, through that of Wells and Harris, to that of Chomsky & Halle. We conclude by offering some suggestions as to why this is not widely recognised in the field.
On the perceptual origin of loanword adaptations: experimental evidence from Japanese
Authors: Peperkamp Sharon , Vendelin Inga , Nakamura Kimihiro
Published: June 2008

Japanese shows an asymmetry in the treatment of word-final [n] in loanwords from English and French: while it is adapted as a moraic nasal consonant in loanwords from English, it is adapted with a following epenthetic vowel in loanwords from French. We provide experimental evidence that this asymmetry is due to phonetic differences in the realisation of word-final [n] in English and French, and, consequently, to the way in which English and French word-final [n] are perceived by native speakers of Japanese. Specifically, French but not English word-final [n] has a strong vocalic release that Japanese listeners perceive as their native vowel [ɯ]. We propose a psycholinguistic model in which most loanword adaptations originate in perceptual assimilation, a process which takes place during perception and which maps non-native sounds and sound structures onto the phonetically closest native ones. We compare our model to alternatives couched within phonological theory.
Subglacial methanogenesis: A potential climatic amplifier?
Authors: Wadham J. L. , Tranter M. , Tulaczyk S. , Sharp M.
Published: June 2008

Subglacial environments are a previously neglected component of the Earth's global carbon cycle, a reflection of the view held until recently that they are dominated by abiotic and oxic conditions. Here we provide a realistic assessment of the theory that the basal regions of the ice sheets that formed over North America and Europe during glaciations were host to significant populations of anaerobic microorganisms, including methanogens, able to metabolize organic carbon sequestered during interglacials and overridden during Quaternary glacials. In doing so, we review the current evidence for subglacial methane release during deglaciation, estimate the size of the subglacial reservoir of organic carbon (SOC), and assess the amount of SOC available to subglacial microbes and the likely pathways and rates of carbon turnover. We then discuss the fate of subglacial methane and the potential impact of its release on atmospheric methane concentrations. We calculate that the SOC equates to 418–610 Pg C and includes carbon from terrestrial soils/vegetation, peatlands, lake, and marine sediments. The SOC that is potentially available for microbial conversion to methane is smaller than this estimate due to (1) glacial erosion, (2) accumulation of recalcitrant organic carbon compounds over time, (3) conversion to carbon dioxide by aerobic/anaerobic respiration, and (4) incomplete conversion of labile organic matter to methane. We estimate that the total SOC available for conversion to methane is 63 Pg C. Our estimates of methane production potentials span a wide range because of the current uncertainty surrounding subglacial metabolic rates. We believe, however, that there is a strong likelihood that subglacial microbes could convert 63 Pg of SOC to methane during a glacial cycle. If this were the case, release of this methane from the ice sheet margins during retreat would need to be episodic in order to significantly impact atmospheric methane concentrations. Our findings suggest that it may well be important to consider subglacial environments as active components of the Earth's carbon cycle. Conclusive determination of the potential impact of subglacial methane production on atmospheric methane concentrations during deglaciation, however, awaits more precise determination of the ability of subglacial microbes to degrade organic carbon components and their associated rates of metabolism under in situ conditions.
Three‐dimensional MHD simulations of Ganymede’s magnetosphere
Authors: Jia Xianzhe , Walker Raymond J. , Kivelson Margaret G. , Khurana Krishan K. , Linker Jon A.
Published: June 2008

Ganymede is unique among planetary moons because it has its own magnetic field strong enough to form a magnetosphere within Jupiter’s magnetospheric environment. Here we report on our three‐dimensional global magnetohydrodynamic (MHD) simulations that model the interaction between Ganymede’s magnetosphere and the corotating Jovian plasma. We use the measured field and particle properties to define our boundary conditions. Our simulations show that, in addition to the familiar structures such as the magnetopause and equatorial current sheet, Ganymede’s magnetosphere extends into an Alfvén wing that mediates the interaction of Ganymede with the plasma and ionosphere of Jupiter. The field‐aligned currents in the Alfvén wing close on themselves not only through the moon and its ionosphere. They also close through the magnetopause and tail current sheets. The pattern of the field‐aligned currents varies according to the orientation of the external magnetic field and asymmetries in the intensities of the parallel currents are organized by the clock angle of the ambient field in the plane perpendicular to the incident flow. The simulations reproduce quite closely the magnetic field structure measured by the Galileo magnetometer for all six close encounters. The magnetopause currents are well resolved in our high resolution simulations, producing sharp rotations in the field orientation consistent with the observations. However, the discrepancies between our model results and the data, such as the weaker field strength near closest approach in multiple simulated flybys, suggest the possibility that Ganymede’s intrinsic magnetic field may be stronger than the accepted value. The magnetosphere produced in our simulations can provide us with realistic estimates of the moon’s magnetic environment thereby enabling us to refine our determination of Ganymede’s internal magnetic field and to better understand the energetic particle behavior.
The evolution of the stratopause during the 2006 major warming: Satellite data and assimilated meteorological analyses
Authors: Manney Gloria L. , Krüger Kirstin , Pawson Steven , Minschwaner Ken , Schwartz Michael J. , Daffer William H. , Livesey Nathaniel J. , Mlynczak Martin G. , Remsberg Ellis E. , Russell James M. , Waters Joe W.
Published: June 2008

Microwave Limb Sounder and Sounding of the Atmosphere with Broadband Emission Radiometry data provide the first opportunity to characterize the four‐dimensional stratopause evolution throughout the life‐cycle of a major stratospheric sudden warming (SSW). The polar stratopause, usually higher than that at midlatitudes, dropped by ∼30 km and warmed during development of a major “wave 1” SSW in January 2006, with accompanying mesospheric cooling. When the polar vortex broke down, the stratopause cooled and became ill‐defined, with a nearly isothermal stratosphere. After the polar vortex started to recover in the upper stratosphere/lower mesosphere (USLM), a cool stratopause reformed above 75 km, then dropped and warmed; both the mesosphere above and the stratosphere below cooled at this time. The polar stratopause remained separated from that at midlatitudes across the core of the polar night jet. In the early stages of the SSW, the strongly tilted (westward with increasing altitude) polar vortex extended into the mesosphere, and enclosed a secondary temperature maximum extending westward and slightly equatorward from the highest altitude part of the polar stratopause over the cool stratopause near the vortex edge. The temperature evolution in the USLM resulted in strongly enhanced radiative cooling in the mesosphere during the recovery from the SSW, but significantly reduced radiative cooling in the upper stratosphere. Assimilated meteorological analyses from the European Centre for Medium‐Range weather Forecasts (ECMWF) and Goddard Earth Observing System Version 5.0.1 (GEOS‐5), which are not constrained by data at polar stratopause altitudes and have model tops near 80 km, could not capture the secondary temperature maximum or the high stratopause after the SSW; they also misrepresent polar temperature structure during and after the stratopause breakdown, leading to large biases in their radiative heating rates. ECMWF analyses represent the stratospheric temperature structure more accurately, suggesting a better representation of vertical motion; GEOS‐5 analyses more faithfully describe stratopause level wind and wave amplitudes. The high‐quality satellite temperature data used here provide the first daily, global, multiannual data sets suitable for assessing and, eventually, improving representation of the USLM in models and assimilation systems.
A regional climate model study of how biomass burning aerosol impacts land‐atmosphere interactions over the Amazon
Authors: Zhang Yan , Fu Rong , Yu Hongbin , Dickinson Robert E. , Juarez Robinson Negron , Chin Mian , Wang Hui
Published: June 2008

Ensemble simulations of a regional climate model assuming smoke aerosol in the Amazon suggest that dynamic changes of cloud cover contributes to the radiative effect of the smoke on the diurnal cycles of surface fluxes and the depth and structure of planetary boundary layer (PBL). In addition to their local effects, the aerosol radiative forcing also appears to weaken or delay the circulation transition from dry to wet season, leading to a weaker moisture transport into the smoke area where the aerosols optical depth, AOD, exceeds 0.3 and a stronger moisture transport and increase of cloudiness in the region upwind to the smoke area. The land surface scheme is modified to improve the regional climate model simulation of the daily mean and diurnal cycle of the surface sensible and latent heat fluxes over the Amazon rain forest. The aerosol radiative forcing is applied to the model during a dry to wet transition season (August–October) in that region. Cloudiness decreases in early afternoon due to the absorption of solar radiation by smoke aerosols partially compensate for the reduction of surface solar flux by aerosol scattering, shifting the strongest changes of surface flux and the PBL to late morning. The reduction of net solar radiation at the surface by smoke is locally largely compensated by reduction of surface sensible flux, with reduction of latent flux only about 30% as large. The strong aerosol absorption in the top 1 km of the aerosol layer stabilizes the 2 to 3 km layer immediately above the daytime PBL and consequently cloudiness decreases. This reduced surface solar flux and more stable lapse rate at the top of the PBL stabilize the lower troposphere. These changes lead to anomalous wind divergence in the southern Amazon and anomalous wind convergence over the equatorial western Amazon in the upwind direction of the smoke area.
Extracellular Diffusion Parameters in the Rat Somatosensory Cortex during Recovery from Transient Global Ischemia/Hypoxia
Authors: Zoremba Norbert , Homola Aleš , Šlais Karel , Voříšek Ivan , Rossaint Rolf , Lehmenkühler Alfred , Syková Eva
Published: June 2008

Changes in the extracellular space diffusion parameters during ischemia are well known, but information about changes during the postischemic period is lacking. Extracellular volume fraction (α) and tortuosity (Λ) were determined in the rat somatosensory cortex using the real-time iontophoretic method; diffusion-weighted magnetic resonance imaging was used to determine the apparent diffusion coefficient of water. Transient ischemia was induced by bilateral common carotid artery clamping for 10 or 15 mins and concomitant ventilation with 6% O2 in N2. In both ischemia groups, a negative DC shift accompanied by increased potassium levels occurred after 1 to 2 mins of ischemia and recovered to preischemic values within 3 to 5 mins of reperfusion. During ischemia of 10 mins duration, α typically decreased to 0.07 ± 0.01, whereas Λ increased to 1.80 ± 0.02. In this group, normal values of α = 0.20 ± 0.01 and Λ = 1.55 ± 0.01 were registered within 5 to 10 mins of reperfusion. After 15 mins of ischemia, α increased within 40 to 50 mins of reperfusion to 0.29 ± 0.03 and remained at this level. Tortuosity (Λ) increased to 1.81 ± 0.02 during ischemia, recovered within 5 to 10 mins of reperfusion, and was increased to 1.62 ± 0.01 at the end of the experiment. The observed changes can affect the diffusion of ions, neurotransmitters, metabolic substances, and drugs in the nervous system.
Role of the p38 Mitogen-Activated Protein Kinase/Cytosolic Phospholipase A2 Signaling Pathway in Blood—Brain Barrier Disruption after Focal Cerebral Ischemia and Reperfusion
Authors: Nito Chikako , Kamada Hiroshi , Endo Hidenori , Niizuma Kuniyasu , Myer D Jeannie , Chan Pak H
Published: June 2008

Cytosolic phospholipase A2 (cPLA2) is a key enzyme that mediates arachidonic acid metabolism, which causes cerebral ischemia-induced oxidative injury, blood—brain barrier (BBB) dysfunction, and edema. Recent reports have shown that p38 mitogen—activated protein kinase (MAPK) is related to phosphorylation and activation of cPLA2 and release of arachidonic acid. However, involvement of the p38 MAPK pathway in cPLA2 activation and of reactive oxygen species in expression of p38 MAPK/cPLA2 after ischemia—reperfusion injury in the brain remains unclear. To address these issues, we used a model of transient focal cerebral ischemia (tFCI) in rats. Western blot analysis showed a significant increase in expression of phospho-p38 MAPK and phospho-cPLA2 in rat brain cortex after tFCI. Activity assays showed that both p38 MAPK and cPLA2 activation markedly increased 1 day after reperfusion. Intraventricular administration of SB203580 significantly suppressed activation and phosphorylation of cPLA2 and attenuated BBB extravasation and subsequent edema. Moreover, overexpression of copper/zinc-superoxide dismutase remarkably diminished activation and phosphorylation of both p38 MAPK and cPLA2 after reperfusion. These findings suggest that the p38 MAPK/cPLA2 pathway may promote BBB disruption with secondary vasogenic edema and that superoxide anions can stimulate this pathway after ischemia—reperfusion injury.
Critical roles of actin-interacting protein 1 in cytokinesis and chemotactic migration of mammalian cells
Authors: Kato Asuka , Kurita Souichi , Hayashi Aya , Kaji Noriko , Ohashi Kazumasa , Mizuno Kensaku
Published: June 2008

Cofilin regulates actin filament dynamics by stimulating actin filament disassembly and plays a critical role in cytokinesis and chemotactic migration. Aip1 (actin-interacting protein 1), also called WDR1 (WD-repeat protein 1), is a highly conserved WD-repeat protein in eukaryotes and promotes cofilin-mediated actin filament disassembly in vitro; however, little is known about the mechanisms by which Aip1 functions in cytokinesis and cell migration in mammalian cells. In the present study, we investigated the roles of Aip1 in cytokinesis and chemotactic migration of human cells by silencing the expression of Aip1 using siRNA (small interfering RNA). Knockdown of Aip1 in HeLa cells increased the percentage of multinucleate cells; this effect was reversed by expression of an active form of cofilin. In Aip1-knockdown cells, the cleavage furrow ingressed normally from anaphase to early telophase; however, an excessive accumulation of actin filaments was observed on the contractile ring in late telophase. These results suggest that Aip1 plays a crucial role in the completion of cytokinesis by promoting cofilin-mediated actin filament disassembly in telophase. We have also shown that Aip1 knockdown significantly suppressed chemokine-induced chemotactic migration of Jurkat T-lymphoma cells, and this was blocked by expression of an active form of cofilin. Whereas control cells mostly formed a single lamellipodium in response to chemokine stimulation, Aip1 knockdown cells abnormally exhibited multiple protrusions around the cells before and after cell stimulation. This indicates that Aip1 plays an important role in directional cell migration by restricting the stimulus-induced membrane protrusion to one direction via promoting cofilin activity.
G-protein binding features and regulation of the RalGDS family member, RGL2
Authors: Ferro Elisa , Magrini David , Guazzi Paolo , Fischer Thomas H. , Pistolesi Sara , Pogni Rebecca , White Gilbert C. , Trabalzini Lorenza
Published: June 2008

RGL2 [RalGDS (Ral guanine nucleotide dissociation stimulator)-like 2] is a member of the RalGDS family that we have previously isolated and characterized as a potential effector for Ras and the Ras analogue Rap1b. The protein shares 89% sequence identity with its mouse orthologue Rlf (RalGDS-like factor). In the present study we further characterized the G-protein-binding features of RGL2 and also demonstrated that RGL2 has guanine-nucleotide-exchange activity toward the small GTPase RalA. We found that RGL2/Rlf properties are well conserved between human and mouse species. Both RGL2 and Rlf have a putative PKA (protein kinase A) phosphorylation site at the C-terminal of the domain that regulates the interaction with small GTPases. We demonstrated that RGL2 is phosphorylated by PKA and phosphorylation reduces the ability of RGL2 to bind H-Ras. As RGL2 and Rlf are unique in the RalGDS family in having a PKA site in the Ras-binding domain, the results of the present study indicate that Ras may distinguish between the different RalGDS family members by their phosphorylation by PKA.
Activation of NADPH oxidase 1 in tumour colon epithelial cells
Authors: Nisimoto Yukio , Tsubouchi Ryoko , Diebold Becky A. , Qiao Shanlou , Ogawa Hisamitsu , Ohara Takuya , Tamura Minoru
Published: June 2008

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22phox, NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP+. The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O2−-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N–Rac1(Q61L)] between truncated NOXA1(1–211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1–292) [C-terminal truncated NOXO1(1–292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O2−-producing activity in Caco-2 cells.
Acellular haemoglobin attenuates hypoxia-inducible factor-1α (HIF-1α) and its target genes in haemodiluted rats
Authors: Manalo Dominador J. , Buehler Paul W. , Baek Jin Hyen , Butt Omer , D'agnillo Felice , Alayash Abdu I.
Published: June 2008

Hb (haemoglobin)-based blood substitutes represent a class of therapeutics designed to correct oxygen deficit under conditions of anaemia and traumatic blood loss. The influences of these agents on HIF-1α (hypoxia-inducible factor-1α) target genes involved in adaptation to hypoxia have so far not been studied. In the study presented here, rats underwent 80% ET (exchange transfusion) with either HS (hetastarch) or a polymerized Hb OG (Oxyglobin®). HS induced dramatic EPO (erythropoietin) gene transcription, reaching a maximum at 4 h post-ET. In contrast, OG suppressed EPO transcription until approx. 24 h post-ET. Large plasma EPO levels that were observed post-ET with HS were significantly blunted in animals transfused with OG. OG, unlike HS, induced a sharp increase in HO-1 (haem oxygenase-1) transcription at 4 h, which declined rapidly within 24 h, whereas modest increases in iNOS [inducible (nitric oxide synthase)] and constitutive NOS [eNOS (endothelial NOS)] were detected over the control. Our results demonstrate for the first time that severe haemodilution-induced erythropoietic responses in kidneys were attenuated by a low-oxygen-affinity cell-free Hb and suggest that tissue-specific oxygen-sensing pathways can be influenced by allosterically modified Hbs.
Modulation of hepatocyte nuclear factor-4α function by the peroxisome-proliferator-activated receptor-γ co-activator-1α in the acute-phase response
Authors: Wang Zhongyan , Burke Peter A.
Published: June 2008

HNF-4α (hepatocyte nuclear factor-4α) is a key regulator of liver-specific gene expression. To understand the mechanisms governing the regulation of HNF-4α function during the APR (acute-phase response), the effects of transcription co-activators, including p300, PGC-1α (peroxisome-proliferator-activated receptor-γ co-activator-1α) and SRC (steroid receptor co-activator)-1α were investigated in an injury cell model. We have shown previously that the HNF-4α-sensitive APR genes ApoB (apolipoprotein B), TTR (transthyretin) and α1-AT (α1-antitrypsin) were regulated at the DNA binding and transcriptional levels after cytokine stimulation. We now show that co-activators have a differential impact on the transactivation of HNF-4α-sensitive genes via HNF-4α-binding sites in ApoB, TTR or α1-AT promoters. PGC-1α strongly enhances the transactivation of ApoB and α1-AT and, to a lesser extent, of TTR, whereas SRC-1α and p300 only have a weak or no effect on these three genes. More importantly, it was found that PGC-1α has a novel role in the modulation of the binding ability of HNF-4α in response to cytokine treatment. Using in vitro and in vivo approaches, electrophoretic mobility-shift and chromatin immunoprecipitation assays, we demonstrate that the reduced HNF-4α–DNA binding ability induced by cytokines is eliminated by overexpression of PGC-1α. Cytokine treatment does not significantly alter the protein levels of HNF-4α and PGC-1α, but it does reduce the recruitment of PGC-1α to HNF-4α-binding sites and thereby decreases transcriptional activity. These results establish the importance of PGC-1α for HNF-4α function and describe a new HNF-4α-dependent regulatory mechanism that is involved in the response to injury.
Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-β-lactamase from Aeromonas hydrophila
Authors: Bebrone Carine , Anne Christine , Kerff Frédéric , Garau Gianpiero , De Vriendt Kris , Lantin Raphaël , Devreese Bart , Van Beeumen Jozef , Dideberg Otto , Frère Jean-Marie , Galleni Moreno
Published: June 2008

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) β-lactamase from Aeromonas hydrophila is a Zn2+-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His118, Asp120, His196 and His263) and in substrate specificity (Val67, Thr157, Lys224 and Lys226), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp120 and His263, and thus is located in the ‘cysteine’ zinc-binding site. His118 and His196 residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val67 plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val67 also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys224 in the binding of substrate. Lys226 is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.
Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases
Authors: Gibellini Federica , Hunter William N. , Smith Terry K.
Published: June 2008

Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (glycerophosphoethanolamine) and GPCho (glycerophosphocholine). Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The Km values for ethanolamine and ATP were found to be 18.4±0.9 and 219±29 μM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a Km 80 times lower than that of ethanolamine. The Km values for choline, ethanolamine and ATP were 31.4±2.6 μM, 2.56±0.31 mM and 20.6±1.96 μM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.
Regulation of a Drosophila melanogaster cGMP-specific phosphodiesterase by prenylation and interaction with a prenyl-binding protein
Authors: Day Jonathan P. , Cleghon Vaughn , Houslay Miles D. , Davies Shireen-A.
Published: June 2008

Post-translational modification by isoprenylation is a pivotal process for the correct functioning of many signalling proteins. The Drosophila melanogaster cGMP-PDE (cGMP-specific phosphodiesterase) DmPDE5/6 possesses a CaaX-box prenylation signal motif, as do several novel cGMP-PDEs from insect and echinoid species (in CaaX, C is cysteine, a is an aliphatic amino acid and X is ‘any’ amino acid). DmPDE5/6 is prenylated in vivo at Cys1128 and is localized to the plasma membrane when expressed in Drosophila S2 cells. Site-directed mutagenesis of the prenylated cysteine residue (C1128S-DmPDE5/6), pharmacological inhibition of prenylation or co-expression of DmPrBP (Drosophila prenyl-binding protein)/δ each alters the subcellular localization of DmPDE5/6. Thus prenylation constitutes a critical post-translational modification of DmPDE5/6 for membrane targeting. Co-immunoprecipitation and subcellular-fractionation experiments have shown that DmPDE5/6 interacts with DmPrBP/δ in Drosophila S2 cells. Transgenic lines allow targeted expression of tagged prenylation-deficient C1128S-DmPDE5/6 in Type I (principal) cells in Drosophila Malpighian tubules, an in vivo model for DmPDE5/6 function. In contrast with wild-type DmPDE5/6, which was exclusively associated with the apical membrane, the C1128S-DmPDE5/6 mutant form was located primarily in the cytosol, although some residual association occurred at the apical membrane. Despite the profound change in intracellular localization of C1128S-DmPDE5/6, active transport of cGMP is affected in the same way as it is by DmPDE5/6. This suggests that, in addition to prenylation and interaction with DmPrBP/δ, further functional membrane-targeting signals exist within DmPDE5/6.
Interaction of tryptophan derivatives with SLC6A14 (ATB0,+) reveals the potential of the transporter as a drug target for cancer chemotherapy
Authors: Karunakaran Senthil , Umapathy Nagavedi S. , Thangaraju Muthusamy , Hatanaka Takahiro , Itagaki Shiro , Munn David H. , Prasad Puttur D. , Ganapathy Vadivel
Published: June 2008

ATB0,+ [SLC6A14 (solute carrier family 6 member 14)] is an Na+/Cl−-coupled amino acid transporter whose expression is upregulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour cells through its ability to inhibit indoleamine dioxygenase. In the present study, we investigated the role of ATB0,+ in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer drug into tumour cells. These studies show that 1-methyltryptophan is a transportable substrate for ATB0,+. The transport process is Na+/Cl−-dependent with an Na+/Cl−/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of α-methyltryptophan as a blocker, not a transportable substrate, for ATB0,+. ATB0,+ can transport 18 of the 20 proteinogenic amino acids. α-Methyltryptophan blocks the transport function of ATB0,+ with an IC50 value of ∼250 μM under conditions simulating normal plasma concentrations of all these 18 amino acids. These results suggest that α-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB0,+ is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB0,+-positive tumour cells with α-methyltryptophan leads to suppression of their colony-forming ability, whereas ATB0,+-negative cell lines are not affected. The blockade of ATB0,+ in these cells with α-methyltryptophan is associated with cell cycle arrest. These studies reveal the potential of ATB0,+ as a drug target for cancer chemotherapy.
Crucial role of the C-terminus of PTEN in antagonizing NEDD4-1-mediated PTEN ubiquitination and degradation
Authors: Wang Xinjiang , Shi Yuji , Wang Junru , Huang Guochang , Jiang Xuejun
Published: June 2008

PTEN (phosphatase and tensin homologue deleted on chromosome 10), a potent tumour suppressor and multifunctional signalling protein, is under intricate regulation. In the present study, we have investigated the mechanism and regulation of PTEN ubiquitination catalysed by NEDD4-1 (neural-precursor-cell-expressed, developmentally down-regulated 4-1), a ubiquitin ligase for PTEN we identified recently. Using the reconstituted assay and cellular analysis, we demonstrated that NEDD4-1-mediated PTEN ubiquitination depends on its intact HECT (homologous to E6-associated protein C-terminus) domain. Instead of using its WW domains (protein–protein interaction domains containing two conserved tryptophan residues) as a protein interaction module, NEDD4-1 interacts with PTEN through its N-terminal region containing a C2 domain as well as the HECT domain. Strikingly, we found that a C-terminal truncated PTEN fragment binds to NEDD4-1 with higher affinity than the full-length PTEN, suggesting an intrinsic inhibitory effect of the PTEN C-terminus on PTEN–NEDD4-1 interaction. Moreover, the C-terminal truncated PTEN is more sensitive to NEDD4-1-mediated ubiquitination and degradation. Therefore the present study reveals that the C-terminus of PTEN plays a critical role in stabilizing PTEN via antagonizing NEDD4-1-induced PTEN protein decay; conversely, truncation of the PTEN C-terminus results in rapid NEDD4-1-mediated PTEN degradation, a possible mechanism accounting for attenuation of PTEN function by certain PTEN mutations in human cancers.
The human receptor tyrosine kinase Axl gene – promoter characterization and regulation of constitutive expression by Sp1, Sp3 and CpG methylation
Authors: Mudduluru Giridhar , Allgayer Heike
Published: June 2008

Axl is a receptor tyrosine kinase which promotes anti-apoptosis, mitogenesis, invasion, angiogenesis and metastasis, and is highly expressed in cancers. However, the transcriptional regulation of this important gene has never been characterized. The present study was initiated to characterize the promoter, cis-acting elements and promoter methylation driving expression of Axl. The 2.4 kb sequence upstream of the translational start site, and sequential 5′-deletions were cloned and revealed a minimal GC-rich region (−556 to +7) to be sufficient for basal Axl promoter activity in Rko, HCT116 and HeLa cells. Within this minimal region, five Sp (specificity protein)-binding sites were identified. Two sites (Sp a and Sp b) proximal to the translation start site were indispensable for Axl promoter activity, whereas mutation of three additional upstream motifs (Sp c, Sp d and Sp e) was of additional relevance. Gel-shift assays and chromatin immunoprecipitation identified that Sp1 and Sp3 bound to all five motifs, and mutation of all motifs abolished binding. Mithramycin, which inhibits binding of Sp factors to GC-rich sites, dramatically reduced Axl promoter activity and Axl, Sp1 and Sp3 expression. In Drosophila Schneider SL2-cells, exogenous expression of Sp1/Sp3 increased Axl promoter activity. Use of Sp1/Sp3 siRNAs (small interfering RNAs) significantly reduced Axl promoter activity and protein levels in Rko and HeLa cells. Methylation-bisulfite sequencing detected methylated CpG sites within three Sp motifs (Sp a, Sp b and Sp c) and GC-rich flanking sequences, and demethylation by 5-aza-2′-deoxycytidine up-regulated Axl and Sp3 expression in low-Axl-expressing Colo206f/WiDr cells, but not in high-Axl-expressing Rko cells. The results of the present study suggest that Axl gene expression in cancer cells is (1) constitutively driven by Sp1/Sp3 bound to five core promoter motifs, and (2) restricted by methylation within/around Sp-binding sites. This might enhance the understanding and treatment of essential mechanisms associated with cancer and other diseases.
CD8+ T-cells and their interaction with other cells in damage to islet β-cells
Authors: Wong F. Susan , Siew L. Khai , Wen Li
Published: June 2008

The autoimmune attack on pancreatic β-cells is orchestrated by a variety of cells that produce cytokines and other toxic mediators. CD8+ T-cells work together with other lymphocytes and antigen-presenting cells to mediate this damage and have been shown in animal models to be important both in the early stages of diabetes development and in the final effector stages. Recently, there has also been much interest in studying CD8+ T-cells that may play a role in human Type 1 diabetes and identifying their antigenic targets. The present paper will focus on the activation of CD8+ T-cells and their interaction with other cells of the immune system and discuss the target antigens and mechanisms of damage that the CD8+ T-cells use in the attack on the islet β-cell.
Macrophages, cytokines and β-cell death in Type 2 diabetes
Authors: Ehses Jan A. , Böni-Schnetzler Marianne , Faulenbach Mirjam , Donath Marc Y.
Published: June 2008

The pathology of islets from patients with Type 2 diabetes displays an inflammatory process characterized by the presence of immune cell infiltration, cytokines, apoptotic cells, amyloid deposits and, eventually, fibrosis. Indeed, analysis of β-cells from patients with Type 2 diabetes displays increased IL-1β (interleukin 1β) expression. Furthermore, increased islet-associated macrophages are observed in human Type 2 diabetic patients and in most animal models of diabetes. Importantly, increased numbers of macrophages are detectable very early in high-fat-fed mice islets, before the onset of diabetes. These immune cells are probably attracted by islet-derived chemokines, produced in response to metabolic stress, and under the control of IL-1β. It follows that modulation of intra-islet inflammatory mediators, particularly interleukin-1β, may prevent islet inflammation in Type 2 diabetes and therefore presents itself as a promising therapeutic approach.
Stem cells and metabolic diseases
Authors: Bernardo Andreia S. , Docherty Kevin
Published: June 2008

Obesity is a metabolic disorder, which has been recognized as a global epidemic. It contributes to insulin resistance, the major cause of Type 2 diabetes, as well as to the development of other related diseases. Our basic premise is that a better understanding of how adult stem cells of the pancreas contribute to the maintenance of the pancreatic β-cell pool against the increased metabolic demands associated with obesity may provide new therapeutic targets for treating diabetes. At the same time, if we knew more about the biology of adipocyte formation, maintenance and deposition in obese individuals, perhaps some control over the adipocyte tissue mass of these individuals would be facilitated and treatment of obesity would become available. Many investigations in the field are therefore aimed at describing how adipocyte stem cells function in the various sites of fat deposition and the extent to which these stem cells contribute to both brown and white adipocytes. Studies on the differentiation of human embryonic stem cells along the pancreatic and adipocyte lineages may therefore better inform approaches to these studies.