bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for the predominance of denitrification as a source of N2O in temperate agricultural soils based on isotopologue measurements
Authors: Opdyke Matthew R. , Ostrom Nathaniel E. , Ostrom Peggy H.
Published: November 2009

Isotopologue values were used to quantify the contribution of denitrification to nitrous oxide (N2O) flux in agricultural and early successional fields in southwest Michigan. Nitrous oxide‐δ15N and δ18O values were poor estimators of microbial origins compared to site preference (SP) (difference in δ15N between the outer and central N atoms of N2O). Site preference was used to evaluate the importance of denitrification (including nitrifier denitrification) in N2O production. Average flux‐weighted SP values for each field ranged between 2.9 and 14.6‰ and, on the basis of SP values for N2O production from denitrification (0‰) and collectively for nitrification and fungal denitrification (37‰), these values indicate that between 61 and 92% of N2O originated from denitrification. Reduction of N2O ranged from undetectable to as much as 50% of production; and because reduction increases SP, our results underestimate the percentage of N2O from bacterial denitrification. The SP values in our study clearly indicate that denitrification is the predominant source of soil‐derived N2O.
Transition to superparamagnetism in chains of magnetosome crystals
Authors: Newell A. J.
Published: November 2009

Magnetotactic bacteria use chains of magnetic crystals to orient them in the Earth's field. Magnetic measurements show that these chains are in a single‐domain state with all the moments pointing along the chain axis. Yet many of the crystals in the chains fall in either the multidomain (MD) or the superparamagnetic (SP) size range for isolated crystals. Magnetostatic coupling between crystals keeps the magnetization uniform and prevents thermally assisted transitions between states. The SP critical size for a chain of magnetite crystals is calculated using a new algorithm. The network of stable states and transition states connecting them is determined using a homotopy continuation method. This determines the energy barriers between stable states, and the critical size can then be calculated using a master equation. As the number of crystals in the chain increases, the SP critical volume approaches a limit that is nearly independent of the shape of the crystals. The cube root of this volume is about 10 nm. Most magnetosome crystal sizes are well above this limit. About half of the magnetosome crystals found in sediments would be MD in isolation. However, there is also a population of cubo‐octahedral crystals, a large fraction of which would be SP in isolation. Such crystals are also formed in fresh water by bacteria in the genusMagnetospirillum. The difference in size and shape between the populations of isometric and nonisometric crystals may be related to redox conditions and the choice of magnetoaerotaxis mechanism.
Scaling relations for seismic cycles on mid‐ocean ridge transform faults
Authors: Boettcher Margaret S. , McGuire Jeffrey J.
Published: November 2009

Mid‐ocean ridge transform faults (RTFs) have thermal structures that vary systematically with tectonic parameters, resulting in predictable seismic characteristics and clear seismic cycles. We develop a scaling relation for repeat time, tR, of the largest expected earthquake, MC: tR = μ−1Δσ2/3CMc1/3AT1/4V−1, where μ is the shear modulus, Δσ is the stress drop, CMc is a constant, AT is the area above 600°C, and V is the slip rate. We identify repeating MC earthquakes by measuring differential arrival times of first orbit Rayleigh waves to determine centroid offsets between pairs of events. Comparing our observations of tR (5–14 years for earthquakes on Gofar and Blanco RTFs) with predictions from our scaling relation, we can constrain RTF stress drops. Specific tests of this scaling relation are proposed for earthquakes on Blanco, Gofar, Discovery, and Clipperton RTFs, which are all expected to have large ruptures in the next few years.
Impact of deforestation on regional surface temperatures and moisture in the Maya lowlands of Guatemala
Authors: Manoharan Vani Starry , Welch Ronald M. , Lawton Robert O.
Published: November 2009

The Petén basin of Guatemala is a region in Mesoamerica experiencing extensive deforestation. Twelve 30 × 30 km2 forested, deforested and partially deforested habitats are selected to compare changes in surface temperature, Normalized Difference Vegetation Index (NDVI) and soil moisture parameters derived from MODIS satellite data during the dry and wet seasons from 2000 to 2008. During the wet season surface temperatures, soil moisture, and NDVI values have similar values in forested and deforested regions. During the dry season, deforested regions tend to be 4–8°C warmer than nearby forested regions, along with significantly lower values of NDVI and soil moisture. From 2000 to 2008, the partially deforested regions became progressively warmer during the dry season, and both NDVI and soil moisture values approached those of the deforested regions. A supervised classification revealed that three partially deforested regions contained 17.7%, 12.9% and 13.4% forests in 2000, decreasing to 2.9%, 8.3% and 4.7%, respectively, in 2008. Overall, partially forested regions in the Petén region decreased in forest cover by 9.3% from 2000 to 2008, while there has been a 0.33% per year deforestation rate over the entire Petén basin.
A study on the low‐latitude daytime E region plasma irregularities using coordinated VHF radar, rocket‐borne, and ionosonde observations
Authors: Patra A. K. , Venkateswara Rao N. , Phanikumar D. V. , Chandra H. , Das U. , Sinha H. S. S. , Pant T. K. , Sripathi S.
Published: November 2009

In this paper we study the off‐electrojet low‐latitude daytime E region plasma irregularities using first multi‐instrument observations in India made during July 2004 by the MST radar from Gadanki (13.5°N, 79.2°E, magnetic latitude 6.4°N), Langmuir probe on board the RH‐300 Mk II rocket, and ionosonde from Sriharikota (13.6°N, 80.2°E, magnetic latitude 6.4°N). Radar echoes were confined to altitudes below 105 km and were observed in the form of a descending echoing layer with the descent rate of 1 km/h. Virtual height of the E layer, as observed by ionosonde, shows identical descending behavior. A detailed analysis based on the radar and ionosonde observations shows that the radar echoes are related to the range spread in the ionogram. Rocket observations made on 23 July 2004 revealed weak plasma irregularities with scale sizes more than 100 m and no noticeable irregularity at shorter scales. The spectral slope of the irregularities observed by the rocket probe is found to be −4 for scales in between 1 km and 100 m. During the rocket launch, radar did not detect any echo conforming that the small‐scale irregularities were not present. Examination of concurrent observations of neutral wind made by TIMED Doppler interferometry suggests that zonal wind plays a crucial role in forming electron density layers, which become unstable via the gradient drift instability with background electric field or/and zonal neutral wind generating low‐latitude E region plasma irregularities.
Observation and analysis of shear instability in the Fraser River estuary
Authors: Tedford E. W. , Carpenter J. R. , Pawlowicz R. , Pieters R. , Lawrence G. A.
Published: November 2009

We investigate the occurrence of shear instability in the Fraser River estuary. Instabilities observed with an echo sounder are compared with a linear stability analysis based on observed velocity and density profiles. We find that each set of observed instabilities coincides with an unstable mode predicted by the Taylor‐Goldstein equation. Each of these instabilities occurs in a region where the gradient Richardson number is less than the critical value of 1/4. Both the Taylor‐Goldstein predictions and the echo soundings indicate the instabilities are concentrated either above or below the density interface. This “one sidedness” is in contrast to the archetypal Kelvin‐Helmholtz instability. Although the dominant source of mixing in the estuary appears to be caused by shear instability, when the tide produces strong near‐bed velocities, small‐scale overturning due to boundary layer turbulence is apparent throughout the depth.
Stratospheric and solar cycle effects on long‐term variability of mesospheric ice clouds
Authors: Lübken F.‐J. , Berger U. , Baumgarten G.
Published: November 2009

Model results of mesospheric ice layers and background conditions at 69°N from 1961 to 2008 are analyzed. The model nudges to European Centre for Medium‐Range Weather Forecasts data below ∼45 km. Greenhouse gas concentrations in the mesosphere are kept constant. At polar mesospheric cloud (PMC) altitudes (83 km) temperatures decrease until the mid 1990s by ‐0.08 K/yr resulting in trends of PMC brightness, occurrence rates, and, to a lesser extent, in PMC altitudes (−0.0166 km/yr). Ice layer trends are consistent with observations by ground‐based and satellite instruments. Water vapor increases at PMC heights and decreases above due to increased freeze‐drying caused by the temperature trend. Temperature trends in the mesosphere mainly come from shrinking of the stratosphere and from dynamical effects. A solar cycle modulation of H2O is observed in the model consistent with satellite observations. The effect on ice layers is reduced because of redistribution of H2O by freeze‐drying. The accidental coincidence of low temperatures and solar cycle minimum in the mid 1990s leads to an overestimation of solar effects on ice layers. A strong correlation between temperatures and PMC altitudes is observed. Applied to historical measurements this gives negligible temperature trends at PMC altitudes (∼0.01–0.02 K/yr). Strong correlations between PMC parameters and background conditions deduced from the model confirm the standard scenario of PMC formation. The PMC sensitivity on temperatures, water vapor, and Ly‐α is investigated. PMC heights show little variation with background parameters whereas brightness and occurrence rates show large variations. None of the background parameters can be ignored regarding its influence on ice layers.
Dynamics and mass balance of Taylor Glacier, Antarctica: 1. Geometry and surface velocities
Authors: Kavanaugh J. L. , Cuffey K. M. , Morse D. L. , Conway H. , Rignot E.
Published: November 2009

Taylor Glacier, Antarctica, exemplifies a little‐studied type of outlet glacier, one that flows slowly through a region of rugged topography and dry climate. This glacier, in addition, connects the East Antarctic Ice Sheet with the McMurdo Dry Valleys, a region much studied for geomorphology, paleoclimate, and ecology. Here we report extensive new measurements of surface velocities, ice thicknesses, and surface elevations, acquired with InSAR, GPS, and GPR. The latter two were used to construct elevation models of the glacier's surface and bed. Ice velocities in 2002–2004 closely matched those in 2000 and the mid‐1970s, indicating negligible interannual variations of flow. Comparing velocities with bed elevations shows that, along much of the glacier, flow concentrates in a narrow axis of relatively fast flowing ice that overlies a bedrock trough. The flow of the glacier over major undulations in its bed can be regarded as a “cascade”; it speeds up over bedrock highs and through valley narrows and slows down over deep basins and in wide spots. This pattern is an expected consequence of mass conservation for a glacier near steady state. Neither theory nor data from this Taylor Glacier study support the alternative view, recently proposed, that an outlet glacier of this type trickles slowly over bedrock highs and flows fastest over deep basins.
Dynamics and mass balance of Taylor Glacier, Antarctica: 2. Force balance and longitudinal coupling
Authors: Kavanaugh J. L. , Cuffey K. M.
Published: November 2009

Taylor Glacier, Antarctica, exemplifies an ice sheet outlet that flows through a region of rugged topography and dry climate. In contrast to other well‐studied outlets, Taylor Glacier moves very slowly, despite a thickness of order 1 km and driving stresses averaging 1.5 bars. Here we analyze new measurements of glacier geometry and surface velocity to elucidate flow dynamics of Taylor Glacier. Force balance and basal temperatures are calculated at six locations along the glacier's length using an algorithm developed for this study. The effects of stress‐gradient coupling on longitudinal flow variations are also examined; we ask whether Kamb and Echelmeyer's (1986) linearized theory adequately describes the observed response of flow to large‐amplitude variations in driving stress. The force balance calculations indicate that no basal motion is needed to explain the observed flow of Taylor Glacier. Inferred basal temperatures are within a few degrees of the melting point in regions of kilometer‐thick ice and well below the melting point elsewhere; deformation of subfreezing ice largely controls the flow of Taylor Glacier. Basal drags are mostly in the range 0.9 to 1.2 bars, and lateral drags are in the range 0.2 to 0.5 bar. Stress‐gradient coupling strongly reduces the variability of velocities along the glacier. The velocity variations can be described as the convolution of a forcing function with a spatial filter, as Kamb and Echelmeyer suggested, but the form of the forcing function differs from the theoretical relation derived for small‐amplitude perturbations (the power on driving stress is one, not three).
How smart is smart?
Authors: Hunter Philip
Published: November 2009

Intelligence likely evolved among social animals and became an important factor for the rise of human civilisation. Philip Hunter follows the debate of how and why human intelligence is still evolving and in which direction.
Agent‐based simulation of T‐cell activation and proliferation within a lymph node
Authors: Bogle Gib , Dunbar P Rod
Published: November 2009

Recent intravital microscopy experiments have revealed the complex behavior of T cells within lymph nodes. Modeling T‐cell responses in lymph nodes now requires integration of cell trafficking and motility with the molecular processes involved in T‐cell activation. We describe an agent‐based model that allows such integration, in which T cells undertake a random walk through a three‐dimensional representation of the lymph node paracortex, integrating signals from dendritic cells (DCs), and proliferating in response. The model accommodates simulation of a large number of T cells packed at realistic densities, and includes dynamic cell trafficking that allows the lymph nodes to swell and shrink as the immune response progresses. The results from the model, including the kinetics of cognate T‐cell proliferation and release, and the changes in their avidity profile, are similar to those observed in vivo. We therefore propose that this modeling framework is capable of successfully simulating T‐cell activation while also accounting for new spatiotemporal knowledge of how T cells and DCs interact. Although some of the parameters used to drive the model are not yet experimentally validated, the model is capable of testing the effects of alternative values for any parameter on the T‐cell response. We intend to refine each aspect of the model in collaboration with both theoreticians and experimentalists.
Study of Echinoids of Gurpi formation, Iran
Authors: Balmaki Behnaz , Asgharian Rostami Masoud
Published: November 2009

In order to detailed study of Echinoids, some samples are collected from Brown limestone member of Seimare from Gurpi Formation with 213 meter thickness which located in the vicinity of Ilam province; settling over Ilam Formation continuously. Paleontological results indicated Campanian - maastrichtian age at this section. Detailed analysis of the 300 samples led to recognition of Bivalve especially Bivalve of Lofa, Brachiopods and five species of Echinoids such as: Salenia nutrix, Globator bleicheri, Orthopsis miliarisi, Goniopygus superbus, and Conulus douville
Theoretical Investigation on Electronic Properties of Topological Materials: Moebius Nanographite *and Conjugated Polymers
Authors: Harigaya Kikuo
Published: November 2009

1. Topological materials: Moebius nanographite and conjugated polymers2. Magnetic domain wall states [1] vs. helical magmetic order in nanographite [2]3. Static polarizability [alpha] and the first hyperpolarizability [beta] are studied, comparing three models: Mobius strip cyclacene, ring cyclacene, and linear strip.4. Opening the knot of the Mobius to form the ring leads to the increase of [alpha] from 268 to 323 a.u. and [beta] increases about three times from 393 to 1049 a.u.5. Opening the ring to form the linear strip, [alpha] increases from 323 to 476 a.u. [beta] as well increases about three times from 1049 to 2814 a.u.[1] K. Wakabayashi and K. Harigaya, J. Phys. Soc. Jpn. 72, 998 (2003).[2] A. Yamashiro, Y. Shimoi, K. Harigaya, and K. Wakabayashi, Physica E 22, 688 (2004).
Measurement of gel characteristics
Authors: Mitchell John
Published: November 2009

In the case of solutions formed from non-gelling hydrocolloids determination of viscosity and viscoelastic properties using rotational rheometers can provide an almost complete description of the rheological properties of interest. Additional information which is sometimes required are extensional viscosities which are very difficult to measure and changes from a particulate system to a “solution” for example starch gelatinisation or dissolution of hydrocolloids. For gels the situation is different. Gel texture is multidimensional. Rotational rheology provides important information but other techniques are required to provide the full picture. This paper covers some of these additional methods. Topics that will be considered are:-

• Difference Between Fundamental and Empirical Rheological Methods• Industrially Important Properties of Gels• Empirical Methods• Fundamental Methods
Small deformation measurements of transient gels using vane rheometry
Authors: Ratcliffe I. , Williams P. A. , English R. J. , Meadows J.
Published: November 2009

Measurement of a model transient gel system, i.e. pre-gelled borate cross-linked konjac glucomannan (KGM) has been facilitated by the use of a controlled stress rheometer in conjunction with a stainless steel vane geometry. Calibration of the vane was undertaken in both shear flow and oscillation and enabled reproduction of data recorded using traditional cone and plate and couette fixtures. The transient nature of the borate cross-links coupled with the sharp blades of teh vane facilitated ‘healing’ of the gel around the geometry, resulting in minimal disturbance to the gel. Alternative techniques employed to measure these gels include in-situ gelation in a parallel plate geometry. Using this method difficulties in achieving sample homogeneity imposed serious restrictions upon the range of polysaccharide and borate concentrations that could be studied, and long in-situ gelation times were unavoidable. The microstructure of borate cross-linked KGM gels has been investigated by small strain oscillation measurements using the vane and the trends in the network relaxation times with variation in both polysaccharide concentration and borate concentration established.
Structural properties of gelatin in mixture with sugar
Authors: Kasapis Stefan , Al-Marhoobi Insaf M. A. , Deszczynski Marcin , Mitchell John R. , Abeysekera Rukmal
Published: November 2009

The structural behaviour of a well-characterized gelatin sample has been revisited to investigate the morphology of its network in the presence of sugar. This was then contrasted with the corresponding properties of the gelling polysaccharides agarose, κ-carrageenan and deacylated gellan reported earlier in the literature. Small deformation dynamic oscillation, large deformation compression testing, differential scanning calorimetry in plain and modulated mode, visual observations and transmission electron microscopy were used to identify the structural characteristics of the biopolymers. Characteristics ranged from the melt and the rubbery plateau through the transition region to the glassy state.

In contrast to the collapse of the polysaccharide gels at intermediate levels of cossolute, gelatin forms reinforced networks. The drop in polysaccharide network strength is accompanied by a decline in the enthalpy of the coil-to-helix transition, whereas the transition enthalpy is more pronounced in gelatin gels in accordance with their strengthening. Tangible evidence of the molecular transformations was obtained using microscopy, with polysaccharides disaggregating and dissolving in the saturated sugar environment. Gelatin, on the other hand, is visualized in an aggregated form thus producing a phase-separated topology with sugar. We feel that these are thought-provoking results for confectionery manufacturers aiming to replace the protein with gelling polysaccharides in product formulations.
Effect of principal ingredients on quality of cookies with dietary fibre
Authors: Piteria M. F. , Nunes C. , Raymundo A. , Sousa I.
Published: November 2009

It is well known that fibre is very important in human diet. Distinct segments of the population, for different reasons, would welcome the existence of ready to use food with high level in fibre.

This work is a contribution to understand the role of fibres in a traditional food – cookies or biscuits. These are also convenient foods, easy to carry pocket food, without any special storage requirements. Fibre is known to be determinant of food structure, namely the insoluble fibre has a strong impact on food texture.

The aim of this work is to optimise cookies formulations, using fibre from different sources (oat, orange and pea). Fibre, fat and cooking time were the independent variables considered. Texture, colour, moisture and water activity were the responses, completed with phase transition temperatures information.

The effects of the various ingredients, interacting together, were studied and the optimum formulations for cookies were determined using a Central Composite Rotatable Experimental Design with five levels for the variables. The glass transition temperature (Tg) was measured by Differential scanning Calorimetry.

This study demonstrates the potential use of dietary fibre as a functional ingredient at levels up to 6 - 8%. The textural characteristics of cookies produced with those levels are more lipids than fibre dependent. This means that those levels of fibre can be used in the cookies base formulations, and still producing cookies within the commercial texture standards.
Focal adhesion kinase‐dependent regulation of adhesive forces involves vinculin recruitment to focal adhesions
Authors: Dumbauld David W. , Michael Kristin E. , Hanks Steven K. , García Andrés J.
Published: November 2009

Background information. FAK (focal adhesion kinase), an essential non‐receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signalling and mechanotransduction. FAK‐dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contribution of FAK to the generation of adhesive forces is not well understood.

Results. Using FAK‐null cells expressing wild‐type and mutant FAK under an inducible tetracycline promoter, we analysed the role of FAK in the generation of steady‐state adhesive forces using micropatterned substrates and a hydrodynamic adhesion assay. FAK expression reduced steady‐state strength by 30% compared with FAK‐null cells. FAK expression reduced VCL (vinculin) localization to focal adhesions by 35% independently of changes in integrin binding and localization of talin and paxillin. RNAi (RNA interference) knock‐down of VCL abrogated the FAK‐dependent differences in adhesive forces. FAK‐dependent changes in VCL localization and adhesive forces were confirmed in human primary fibroblasts with FAK knocked down by RNAi. The autophosphorylation Tyr‐397 and kinase domain Tyr‐576/Tyr‐577 sites were differentially required for FAK‐mediated adhesive responses.

Conclusions. We demonstrate that FAK reduces steady‐state adhesion strength by modulating VCL recruitment to focal adhesions. These findings provide insights into the role of FAK in mechanical interactions between a cell and the extracellular matrix.
Role of lipid metabolism in hepatitis C virus assembly and entry
Authors: Popescu Costin‐Ioan , Dubuisson Jean
Published: November 2009

HCV (hepatitis C virus) represents a major global health problem. A consistent body of evidence has been accumulating, suggesting a peculiar overlap between the HCV life cycle and lipid metabolism. This association becomes evident both for the clinical symptoms of HCV infection and the molecular mechanisms underlying the morphogenesis and entry process of this virus. The HCV core–lipid droplets association seems to be central to the HCV morphogenesis process. Moreover, the biogenesis pathway of very‐low‐density lipoproteins has been shown to be involved in HCV morphogenesis with MTP (microsomal triacylglycerol transfer protein), ApoB (apolipoprotein B) and ApoE (apolipoprotein E) as essential elements in the production of infectious HCV particles. HCV infectivity also correlates with the lipidation status of the particles. Furthermore, some HCV cellular receptors and the regulation of the entry process are also connected to lipoproteins and lipid metabolism. Specifically, lipoproteins modulate the entry process and the cholesterol transporter SR‐BI (scavenger receptor class B type I) is a cellular entry factor for HCV. The present review aims to summarize the advances in our understanding of the HCV–lipid metabolism association, which may open new therapeutic avenues.
Development of an intracellularly acting inhibitory peptide selective for PKN
Authors: Shiga Kazuhiro , Takayama Kentaro , Futaki Shiroh , Hutti Jessica E. , Cantley Lewis C. , Ueki Katsuko , Ono Yoshitaka , Mukai Hideyuki
Published: November 2009

PKNs form a subfamily of the AGC serine/threonine protein kinases, and have a catalytic domain homologous with that of PKC (protein kinase C) in the C-terminal region and three characteristic ACC (antiparallel coiled-coil) domain repeats in the N-terminal region. The preferred peptide phosphorylation motif for PKNs determined by a combinatorial peptide library method was highly similar to that of PKCs within a 10-amino-acid stretch. Previously reported PKN inhibitory compounds also inhibit PKCs to a similar extent, and no PKN selective inhibitors have been commercially available. We have identified a 15-amino-acid peptide inhibitor of PKNs based on amino acids 485–499 of the C-terminal region of the C2-like domain of PKN1. This peptide, designated as PRL, selectively inhibits the kinase activity of all isoforms of PKN (Ki=0.7 μM) towards a peptide substrate, as well as autophosphorylation activity of PKN in vitro, in contrast with PKC. Reversible conjugation by a disulfide bond of a carrier peptide bearing a penetration accelerating sequence to PRL, facilitated the cellular uptake of this peptide and significantly inhibited phosphorylation of tau by PKN1 at the PKN1-specific phosphorylation site in vivo. This peptide may serve as a valuable tool for investigating PKN activation and PKN-mediated responses.
Characterization of the haem oxygenase protein family in Arabidopsis thaliana reveals a diversity of functions
Authors: Gisk Bjoern , Yasui Yukiko , Kohchi Takayuki , Frankenberg-Dinkel Nicole
Published: November 2009

HOs (haem oxygenases) catalyse the oxidative cleavage of haem to BV (biliverdin), iron and carbon monoxide. In plants, the product of the reaction is BV IXα, the precursor of the PHY (phytochrome) chromophore and is thus essential for proper photomorphogenesis. Arabidopsis thaliana contains one major biochemically characterized HO (HY1) and three additional putative HOs (HO2, HO3 and HO4). All four proteins are encoded in the nucleus but contain chloroplast translocation sequences at their N-termini. The transit peptides of all four proteins are sufficient for chloroplast translocalization as shown by GFP (green fluorescent protein) reporter gene fusions. Overall, all four proteins can be divided into two subfamilies: HO1 and HO2. Here we show that all members of the HO1 subfamily (HY1, HO3 and HO4) are active monomeric HOs and can convert haem to BV IXα using spinach Fd (ferredoxin) as an electron donor. Addition of a second electron donor, such as ascorbate, led to a 10-fold increase in the haem conversion rate. Furthermore, haem turnover is also promoted by light when spinach thylakoids are present. All HO1 family members displayed similar kinetic parameters indicating they all have a possible involvement in PHY chromophore biosynthesis. HO2 did not yield sufficient amounts of soluble protein and therefore required the construction of a synthetic gene adapted to the codon usage of Escherichia coli. HO2 is unable to bind or degrade haem and therefore it is not a haem oxygenase. However, HO2 shows strong binding of proto IX (protoporphyrin IX), a precursor for both haem and chlorophyll biosynthesis. A possible function of HO2 in the regulation of tetrapyrrole metabolism is discussed.
PpoC from Aspergillus nidulans is a fusion protein with only one active haem
Authors: Brodhun Florian , Schneider Stefan , Göbel Cornelia , Hornung Ellen , Feussner Ivo
Published: November 2009

In Aspergillus nidulans Ppos [psi (precocious sexual inducer)-producing oxygenases] are required for the production of so-called psi factors, compounds that control the balance between the sexual and asexual life cycle of the fungus. The genome of A. nidulans harbours three different ppo genes: ppoA, ppoB and ppoC. For all three enzymes two different haem-containing domains are predicted: a fatty acid haem peroxidase/dioxygenase domain in the N-terminal region and a P450 haem-thiolate domain in the C-terminal region. Whereas PpoA was shown to use both haem domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8-hydroperoxide isomerization), we found that PpoC apparently only harbours a functional haem peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyses mainly the dioxygenation of linoleic acid (18:2Δ9Z,12Z), yielding 10-HPODE (10-hydroperoxyoctadecadienoic acid). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-KODE (10-keto-octadecadienoic acid) and 10-HODE (10-hydroxyoctadecadienoic acid). In parallel, decomposition of 10-HPODE into 10-ODA (10-octadecynoic acid) and volatile C-8 alcohols that are, among other things, responsible for the characteristic mushroom flavour. Besides these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE into the respective epoxy alcohols: 12,13-epoxy-8-HOME (where HOME is hydroxyoctadecenoic acid) and 12,13-epoxy-10-HOME. By using site-directed mutagenesis we demonstrated that both enzymes share a similar mechanism for the oxidation of 18:2Δ9Z,12Z; they both use a conserved tyrosine residue for catalysis and the directed oxygenation at the C-8 and C-10 is most likely controlled by conserved valine/leucine residues in the dioxygenase domain.
The adaptor protein EBP50 is important for localization of the protein kinase A–Ezrin complex in T-cells and the immunomodulating effect of cAMP
Authors: Stokka Anne Jorunn , Mosenden Randi , Ruppelt Anja , Lygren Birgitte , Taskén Kjetil
Published: November 2009

We recently reported that the dual-specificity AKAP (A-kinaseanchoring protein) Ezrin targets type I PKA (protein kinase A) to the vicinity of the TCR (T-cell receptor) in T-cells and, together with PAG (phosphoprotein associated with glycosphingolipid-enriched membrane microdomains) and EBP50 [ERM (Ezrin/Radixin/Moesin)-binding phosphoprotein 50], forms a scaffold that positions PKA close to its substrate, Csk (C-terminal Src kinase). This complex is important for controlling the activation state of T-cells. Ezrin binds the adaptor protein EBP50, which again contacts PAG. In the present study, we show that Ezrin and EBP50 interact with high affinity (KD=58±7 nM). A peptide corresponding to the EB (Ezrin-binding) region in EBP50 (EBP50pep) was used to further characterize the binding kinetics and compete the Ezrin–EBP50 interaction by various methods in vitro. Importantly, loading T-cells with EBP50pep delocalized Ezrin, but not EBP50. Furthermore, disruption of this complex interfered with cAMP modulation of T-cell activation, which is seen as a reversal of cAMP-mediated inhibition of IL-2 (interleukin 2) production, demonstrating an important role of EBP50 in this complex. In summary, both the biochemical and functional data indicate that targeting the Ezrin–EBP interaction could be a novel and potent strategy for immunomodulation.
Chicken ileal bile-acid-binding protein: a promising target of investigation to understand binding co-operativity across the protein family
Authors: Guariento Mara , Assfalg Michael , Zanzoni Serena , Fessas Dimitrios , Longhi Renato , Molinari Henriette
Published: November 2009

Protein–bile acid interactions are crucial microscopic events at the basis of both physiological and pathological biochemical pathways. BABPs (bile-acid-binding proteins) are intracellular transporters able to bind ligands with different stoichiometry, selectivity and co-operativity. The molecular determinants and energetics of interaction are the observables that connect the microscopic to the macroscopic frameworks. The present paper addresses the study and proposes a mechanism for the multi-site interaction of bile acids with chicken I-BABP (ileal BABP) with the aim of elucidating the determinants of ligand binding in comparison with homologous proteins from different species and tissues. A thermodynamic binding model describing two independent consecutive binding sites is derived from isothermal titration calorimetry experiments and validated on the basis of both protein-observed and ligand-observed NMR titration data. It emerges that a singly bound protein is relatively abundant at low ligand/protein molar ratios assessing the absence of strong co-operativity. Both the measured energetics of binding and the distributed protein chemical-shift perturbations are in agreement with a first binding event triggering a global structural rearrangement. The enthalpic and entropic contributions associated with binding of the first ligand indicate that the interaction increases stability and order of the bound protein. The results described in the present study point to the presence of a protein scaffold which is able to establish long-range communication networks, but does not manifest positive-binding co-operativity, as observed for the human protein. We consider chicken I-BABP a suitable model to address the molecular basis for a gain-of-function on going from non-mammalian to mammalian species.
Peroxisome-proliferator-activated receptor-binding protein (PBP) is essential for the growth of active Notch4-immortalized mammary epithelial cells by activating SOX10 expression
Authors: Zhu Yiwei Tony , Jia Yuzhi , Hu Liping , Qi Chao , Prasad Megana K. , McCallion Andrew S. , Zhu Yi-Jun
Published: November 2009

PBP (peroxisome-proliferator-activated receptor-binding protein) [Med1 (mediator 1)/TRAP220 (thyroid-hormone-receptor-associated protein 220)] is essential for mammary gland development. We established a mammary epithelial cell line with a genotype of PBPLoxP/LoxP by expressing an active form of Notch4. Null mutation of PBP caused severe growth inhibition of the Notch4-immortalized mammary cells. We found that truncated PBP without the two LXXLL motifs could reverse the growth inhibition due to the deficiency of endogenous PBP, indicating that signalling through nuclear receptors is unlikely to be responsible for the growth inhibition as the result of PBP deficiency. Loss of PBP expression was shown to completely ablate the expression of SOX10 [Sry-related HMG (high-mobility group) box gene 10]. The re-expression of SOX10 was capable of reversing the growth inhibition due to PBP deficiency, whereas suppressed expression of SOX10 inhibited the growth of Notch4-immortalized mammary cells. Further studies revealed PBP is directly recruited to the enhancer of the SOX10 gene, indicating that SOX10 is a direct target gene of PBP. We conclude that PBP is essential for the growth of Notch4-immortalized mammary cells by activating SOX10 expression, providing a potential molecular mechanism through which PBP regulates the growth of mammary stem/progenitor cells.
Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface
Authors: Wenzel Sabine , Martins Berta M. , Rösch Paul , Wöhrl Birgitta M.
Published: November 2009

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176–273) at a resolution of 1.55 Å (1 Å=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE” appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.
Cell death and lumen formation in spheroids of MCF‐7 cells
Authors: Amaral Do Bussador Jônatas , Urabayashi Shiniti Marcel , Machado‐Santelli Maria Gláucia
Published: November 2009

3D (three‐dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F‐actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF‐7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.
β‐Adrenergic‐induced CD40 overexpression on gingival fibroblasts: Role of PGE2
Authors: Furlán César , Sterin‐Borda Leonor , Borda Enri
Published: November 2009

CD40, a member of the tumour necrosis factor‐α receptor family, is constitutively expressed by cells of haematopoietic and non‐haematopoietic origin, including fibroblasts. Signalling through this receptor molecule regulates inflammatory mediator secretion by many cell types. The work has been performed in healthy subjects and the authors studied, by cellular culture, flow cytometric analysis and ELISA assay, the expression of CD40 and PGE2(prostaglandin E2) generation on gingival fibroblasts stimulated by β‐AR (β‐adrenoceptor) agonists. Herein, the authors demonstrate that β‐AR subtype activation via their own specific agonists markedly increased CD40 expression on human gingival fibroblasts. This effect was prevented by β‐AR subtype‐specific antagonists. In addition, gingival fibroblast β‐AR stimulation resulted in an increase in PGE2generation. The inhibition of PLA2(phospholipase A2) and COX‐1 (cyclo‐oxygenase‐1) but not COX‐2 impaired β‐AR increase of PGE2, an effect that was restored by the addition of low concentrations of PGE2, suggesting that PGE2generation is implicated in the mechanism underlying β‐AR‐agonist‐mediated CD40 overexpression. Our work has revealed an endogenous β‐AR mediator network involving gingival fibroblasts.
A mutation in β‐tubulin and a sustained dependence on androgen receptor signalling in a newly established docetaxel‐resistant prostate cancer cell line
Authors: Hara Takahito , Ushio Kazutaka , Nishiwaki Mayumi , Kouno Jin , Araki Hideo , Hikichi Yukiko , Hattori Masahiko , Imai Yumi , Yamaoka Masuo
Published: November 2009

The mechanisms of docetaxel resistance in PC (prostate cancer) are unclear because of the lack of suitable experimental models, and no effective treatment exists for docetaxel‐resistant PC. We established a docetaxel‐resistant cell line, LNDCr, from an androgen‐refractory PC cell line, LNCaP‐hr, by intermittent exposure to docetaxel in vitro. The LNDCr cells harboured an F270I mutation in class I β‐tubulin, and demonstrated impaired tubulin polymerization by docetaxel. AR signalling was sustained in LNDCr cells, and AR knockdown suppressed the growth of LNDCr cells. These results suggest that an acquired mutation in β‐tubulin is associated with docetaxel resistance in PC and that a novel AR‐targeted therapy is effective for docetaxel‐resistant PC.
Porcine proximal tubular cells (LLC‐PK1) are able to tolerate high levels of lithium chloridein vitro: assessment of the influence of 1–20 mM LiCl on cell death and alterations in cell biology and biochemistry
Authors: Lucas Kirsten C. , Hart David A. , Becker Rolf W.
Published: November 2009

Lithium, a prophylactic drug for the treatment of bipolar disorder, is prescribed with caution due to its side effects, including renal damage. In this study porcine LLC‐PK1renal tubular cells were used to establish the direct toxicity of lithium on proximal cells and gain insights into the molecular mechanisms involved. In the presence of LiCl, cell proliferation exhibited insignificant decreases in a concentration‐dependent manner, but once confluent, constant cell numbers were observed. Cell cycle studies indicated a small dose‐dependant accumulation of cells in the G2/M stage after 24 h, as well as an increase in cells in the G0/G1phase after treatment with 1–10 mM LiCl, but not at 20 mM LiCl. No evidence of apoptosis was observed based on cell morphology or DNA fragmentation studies, or evidence of protein expression changes for Bax, Bcl‐2, and p53 proteins using immunocytochemistry. In addition caspases 3, 8 and 9 activity remained unaltered between control and lithium‐treated cultures. To conclude, exposure to high concentrations of lithium did not result in overt toxic effects to LLC‐PK1renal cells, although LiCl did alter some aspects of cell behaviour, which could potentially influence function over time.
Characterization of MSCs from human placental decidua basalis in hypoxia and serum deprivation
Authors: Huang Yong‐Can , Yang Zhi‐Ming , Jiang Neng‐Gang , Chen Xiao‐He , Li Xiu‐Qun , Tan Mei‐Yun , Zhou Kun‐Peng , Tang Li , Xie Hui‐Qi , Deng Li
Published: November 2009

Recently, we reported that human PDB (placental decidua basalis) is an excellent source of MSCs (mesenchymal stem cells), meanwhile, PDB‐MSCs could survive under hypoxia and serum deprivation. Herein, we investigated the proliferation, clonogentic efficiency, phenotypes, metabolic activity and cytokines secretion of PDB‐MSCs in hypoxia and serum deprivation. PDB‐MSCs were cultured in four groups: normoxia (20% O2) and complete medium [10% FBS (foetal bovine serum)+DMEM‐HG (Dulbecco's modified Eagle's medium‐high glucose)], hypoxia and complete medium, normoxia and serum deprivation (0% FBS), and hypoxia and serum deprivation. After 96 h of culture in the above groups, PDB‐MSCs maintain the phenotypes stably. Interestingly, hypoxia notably enhanced the proliferation, colony‐forming potential and lactate/glucose ratio in complete medium, but suppressed the secretion of BMP‐2 (bone morphogenetic protein‐2) and bFGF (basic fibroblast growth factor), while it did not change the quantity of VEGF (vascular endothelial growth factor) and bFGF in serum deprivation. Although PDB‐MSCs grew slowly and seldom formed a colony unit in hypoxia and serum deprivation, they possessed a moderate metabolism. In conclusion, our results indicate that PDB‐MSCs appear to be promising seed cells for ischaemia‐related tissue engineering.
Distinct localization of histone H3 methylation in the vegetative nucleus of lily pollen
Authors: Sano Yaeko , Tanaka Ichiro
Published: November 2009

We analysed the distribution of histone H3 modifications in the nucleus of the vegetative cell (the vegetative nucleus) during pollen development in lily (Lilium longiflorum). Among the modifications specifically and/or abundantly present in the vegetative nucleus, dimethylation of histone H3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) were found in heterochromatin, whereas trimethylation of histone H3 at lysine 27 (H3K27me3) was localized in euchromatin in the vegetative nucleus. Such unique localization of the histone H3 methylation marks, particularly of H3K27me3, within a nucleus was not observed in lily nuclei other than the vegetative nucleus. The level of H3K27me3 increased in the euchromatic region of the vegetative nucleus during pollen maturation. The results suggest that H3K27me3 controls the gene expression of the vegetative cell during pollen maturation.