bluuebunny/crossref_metadata_2025 huggingface dataset:
Physics of electron mobility independent of channel orientation in n-channel transistors based on (100) silicon wafers and its experimental verification
Authors: Lau W. S. , Yang Peizhen , Chen T. P. , Siah S. Y. , Chan L.
Published: October 2010

The six degenerate ellipsoid model can be used to study the effects of channel orientation on electron mobility. However, this approach has two assumptions: (i) the conduction band minimum near the interface between gate dielectric and silicon channel is the same as that of bulk silicon, (ii) the momentum relaxation time is independent of the channel orientation. This letter shows that the effective conductivity electron mass of (100) surface-oriented silicon is independent of channel orientation even though the actual conduction band minimum may be slightly different from the six degenerate ellipsoid model. Experimental data are provided to support our theory.
Indium segregation in AlInN/AlN/GaN heterostructures
Authors: Minj A. , Cavalcoli D. , Cavallini A.
Published: October 2010

AlInN/AlN/GaN heterostructures were characterized by atomic force microscopy. V-defects and channels were observed. In phase-contrast mode, these features were found related to inhomogeneities associated with In-segregation (and/or In-diffusion) and Al-rich surface reconstruction. The electrical characterization via conductive atomic force microscopy showed enhanced conductivity regions related to In-rich traces within channels and V-defects.
Paramagnetism in Mn/Fe implanted ZnO
Authors: Gunnlaugsson H. P. , Mølholt T. E. , Mantovan R. , Masenda H. , Naidoo D. , Dlamini W. B. , Sielemann R. , Bharuth-Ram K. , Weyer G. , Johnston K. , Langouche G. , Ólafsson S. , Gíslason H. P. , Kobayashi Y. , Yoshida Y. , Fanciulli M. ,   ISOLDE Collaboration
Published: October 2010

Prompted by the generally poor understanding of the nature of magnetic phenomena in 3d-metal doped ZnO, we have undertaken on-line F57e Mössbauer spectroscopy on ZnO single crystals in an external magnetic field of 0.6 T, following the implantation of radioactive M57n ions at room temperature. The Mössbauer spectra of the dilute Fe impurities are dominated by sextets whose angular dependence rules out an ordered magnetic state (which had been previously proposed) but are well accounted for on the basis of Fe3+ paramagnetic centers on substitutional Zn sites with unusually long relaxation times (>20 ns).
Superiority of common-base to common-emitter heterojunction bipolar transistors
Authors: Qin Guoxuan , Wang Guogong , McCaughan Leon , Ma Zhenqiang
Published: October 2010

Common-emitter (CE) configuration of bipolar junction transistors has been used in virtually all amplifications since the invention of transistor, whereas common-base (CB) configuration has been rarely used due to its inferior performance in comparison to CE. For heterojunction bipolar transistors (HBTs) this conviction needs to be changed. We compared the radio-frequency (rf) power handling capability of the HBT between CE and CB configurations and analyzed their amplification mechanisms. It is found that CB HBT significantly outperforms CE HBT under proper bias conditions, revealing the significant superiority of CB to CE configuration of HBTs for rf power amplification.
Improvement in open circuit voltage of organic solar cells by inserting a thin phosphorescent iridium complex layer
Authors: Huang Jiang , Yu Junsheng , Guan Zhiqiang , Jiang Yadong
Published: October 2010

The authors develop organic solar cells (OSCs) with a multicharge separation (MCS) interface by inserting ∼4 nm thin layer of phosphorescent bis(2-(4-tertbutylphenyl) benzothiazolato-N,C2,) iridium (acetylacetonate) (t-bt)2Ir(acac) comparing with copper phthalocyanine (CuPc) between pentacene/C60 heterojunction. The result showed that open circuit voltage is remarkably enhanced without obviously changing external quantum efficiency ηEQE and short-circuit current density. As a result, power conversion efficiency is improved from 1.53% for pentacene/C60 device to 1.83% with sandwich layer (t-bt)2Ir(acac) and 1.98% with CuPc. Moreover, a suitable equivalent circuit model is proposed to intuitively reveal the inner photogeneration process of OSC with MCS interface.
Delocalized-localized transition in a semiconductor two-dimensional honeycomb lattice
Authors: De Simoni G. , Singha A. , Gibertini M. , Karmakar B. , Polini M. , Piazza V. , Pfeiffer L. N. , West K. W. , Beltram F. , Pellegrini V.
Published: October 2010

We report the magnetotransport properties of a two-dimensional electron gas in a modulation-doped AlGaAs/GaAs heterostructure subjected to a lateral potential with honeycomb geometry. Periodic oscillations of the magnetoresistance and a delocalized-localized transition are shown by applying a gate voltage. We argue that electrons in such artificial-graphene lattices offer a promising approach for the simulation of quantum phases dictated by Coulomb interactions.
Surface plasmon beam splitting by the photon tunneling through the plasmonic nanogap
Authors: Lee Seung-Yeol , Park Junghyun , Woo Inchul , Park Namkyoo , Lee Byoungho
Published: October 2010

A compact device that splits a surface plasmon beam by using double triangular prisms coated on a metal surface is proposed. Due to the photon tunneling effect of the evanescent plasmon mode between the two plasmonic prisms, the incident plasmon beam from one plasmonic prism can be partially transmitted to the other, resulting in the splitting of the plasmon beam. Theoretical study and simulation results for the power dividing ratio of the device are in good agreement with the experimental demonstration. It is believed that the results show their great potential for the realization of the plasmonic interferometer system.
The customized reflections of freeform mirrors
Authors: Hicks R. Andrew
Published: October 2010

Light that reflects off mirrors that have carefully calculated and machined shapes can produce images with surprising and useful properties.
Shaping optical space with metamaterials
Authors: Wegener Martin , Linden Stefan
Published: October 2010

By controlling the local electric and magnetic properties of a material, researchers can tailor the flow of light and create exotic optical devices.
The Excited Triplet State Properties of Titanyl Phthalocyanine and its Sulfonated Derivatives
Authors: Zhang Xian-Fu , Huang Jingyao , Xi Qian , Wang Yun
Published: October 2010

Titanyl phthalocyanine (TiOPc) is a well-known, excellent photoconductive material for laser printers and photocopying machines. Its organic derivatives have recently been shown to be excellent photosensitizers for singlet oxygen [O2(1Δg)] production. The excited triplet state properties of TiOPc, in homogeneous DMSO solution, were measured in this study for the first time by nanosecond laser flash photolysis. The data enabled comparisons to be drawn with TiOPcS4 and zinc phthalocyanine (ZnPc), ultimately providing a better understanding of the reported observations. Absorption, fluorescence, and O2(1Δg) sensitization were also studied. TiOPcS4 in DMSO shows remarkably different fluorescence properties from that reported in aqueous solution: both the fluorescence quantum yield (Φf = 0.068) and the fluorescence lifetime (τf = 3.71 ns) were much larger than that reported for aqueous solutions (0.012 and 0.09 ns, respectively). The photosensitizing properties of TiOPcS4 in DMSO are also so significantly better than that in aqueous solution, i.e. triplet lifetime (τT) of 252 μs, triplet quantum yield (ΦT) of 0.42, and the quantum yield of O2(1Δg) (ΦΔ) of 0.49; compare with values of 60 μs, 0.32, 0.13 reported in aqueous solution. TiOPc, however, shows comparable photophysical properties to that of ZnPc, a well-recognized photosensitizer. These results suggest that TiOPc and its derivatives are not only good photoconductors but also good photosensitizers of O2(1Δg), which may find application in photodynamic therapies for treatment of cancer.
Approaches to the Synthesis of the Galbulimima Alkaloid Himandrine
Authors: O'Connor Patrick D. , Del Signore Giuseppe , McLachlan Matthew M. W. , Willis Anthony C. , Mander Lewis N.
Published: October 2010

The hexacyclic skeleton of himandrine (2), which is present in 15 of the more complex alkaloids obtained from the bark of the tropical rain forest tree Galbulimima belgraveana has been prepared by means of a 19-step synthesis beginning with the known [3.2.1]-benzobicyclooctene intermediate 9. An alternative approach is also described, thus far culminating in 34. Key transformations include Diels–Alder cycloadditions, ring contractions, a Curtius rearrangement, a Birch reduction, an intramolecular nucleophilic amination, and a palladium-mediated alkene amination.
Identification of apolipoprotein C-I in rainbow trout seminal plasma
Authors: Nynca Joanna , Dietrich Mariola A. , Karol Halina , Ciereszko Andrzej
Published: October 2010

Three distinct bands with high electrophoretic migration rates were isolated and purified from rainbow trout seminal plasma. The molecular masses of these bands were determined to be 5158.8, 4065.9 and 4929.0 Da. The N-terminal amino acids sequences were elucidated and were found to have high homology with Atlantic salmon apolipoprotein C-I. It can be concluded that apolipoprotein C-I is a major component of rainbow trout seminal plasma. Further studies are necessary to confirm the protective effects of apolipoprotein C-I on spermatozoa in terms of the stabilisation of the sperm membrane.
The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy
Authors: Di Bartolomeo Sabrina , Corazzari Marco , Nazio Francesca , Oliverio Serafina , Lisi Gaia , Antonioli Manuela , Pagliarini Vittoria , Matteoni Silvia , Fuoco Claudia , Giunta Luigi , D'Amelio Marcello , Nardacci Roberta , Romagnoli Alessandra , Piacentini Mauro , Cecconi Francesco , Fimia Gian Maria
Published: October 2010

Autophagy is an evolutionary conserved catabolic process involved in several physiological and pathological processes such as cancer and neurodegeneration. Autophagy initiation signaling requires both the ULK1 kinase and the BECLIN 1–VPS34 core complex to generate autophagosomes, double-membraned vesicles that transfer cellular contents to lysosomes. In this study, we show that the BECLIN 1–VPS34 complex is tethered to the cytoskeleton through an interaction between the BECLIN 1–interacting protein AMBRA1 and dynein light chains 1/2. When autophagy is induced, ULK1 phosphorylates AMBRA1, releasing the autophagy core complex from dynein. Its subsequent relocalization to the endoplasmic reticulum enables autophagosome nucleation. Therefore, AMBRA1 constitutes a direct regulatory link between ULK1 and BECLIN 1–VPS34, which is required for core complex positioning and activity within the cell. Moreover, our results demonstrate that in addition to a function for microtubules in mediating autophagosome transport, there is a strict and regulatory relationship between cytoskeleton dynamics and autophagosome formation.
BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair
Authors: Ismail Ismail Hassan , Andrin Christi , McDonald Darin , Hendzel Michael J.
Published: October 2010

Polycomb group (PcG) proteins are major determinants of cell identity, stem cell pluripotency, and epigenetic gene silencing during development. The polycomb repressive complex 1, which contains BMI1, RING1, and RING2, functions as an E3-ubuiquitin ligase. We found that BMI1 and RING2 are recruited to sites of DNA double-strand breaks (DSBs) where they contribute to the ubiquitylation of γ-H2AX. In the absence of BMI1, several proteins dependent on ubiquitin signaling, including 53BP1, BRCA1, and RAP80, are impaired in recruitment to DSBs. Loss of BMI1 sensitizes cells to ionizing radiation to the same extent as loss of RNF8. The simultaneous depletion of both proteins revealed an additive increase in radiation sensitivity. These data uncover an unexpected link between the polycomb and the DNA damage response pathways, and suggest a novel function for BMI1 in maintaining genomic stability.
Noise in the World
Authors: Bogen Jim
Published: October 2010

This essay uses Györgi Buzsáki's use of EEG data to draw conclusions about brain function as an example to show that investigators sometimes draw conclusions from noisy data by analyzing the noise rather than by extracting a signal from it. The example makes vivid some important differences between McAllister's, Woodward's, and my ideas about how data are interpreted.
Kuhn on Essentialism and the Causal Theory of Reference
Authors: Kuukkanen Jouni-Matti
Published: October 2010

The causal theory of reference is often taken to provide a solution to the problems, such as incomparability and referential discontinuity, that the meaning-change thesis raised. I show that Kuhn successfully questioned the causal theory and Putnam's idea that reference is determined via the sameness relation of essences that holds between a sample and other members of a kind in all possible worlds. Putnam's single ‘essential’ properties may be necessary but not sufficient to determine membership in a kind category. Kuhn argued that extension is fixed by similarity-dissimilarity relations that are liable to change in taxonomic reorganizations of science.
Modelling of Kelvin–Helmholtz instability and splashing of melt layers from plasma-facing components in tokamaks under plasma impact
Authors: Miloshevsky G.V. , Hassanein A.
Published: October 2010

Plasma-facing components (PFCs) in tokamaks are exposed to high-heat loads during abnormal events such as plasma disruptions and edge-localized modes. The most significant erosion and plasma contamination problem is macroscopic melt splashes and losses from metallic divertor plates and wall materials into core plasma. The classical linear stability analysis is used to assess the initial conditions for development and growth of surface waves at the plasma–liquid metal interface. The maximum velocity difference and critical wavelengths are predicted. The effects of plasma density, surface tension and magnetic field on the stability of plasma–liquid tungsten flows are analytically investigated. The numerical modelling predicts that macroscopic motion and melt-layer losses involve the onset of disturbances on the surface of the tungsten melt layer with relatively long wavelengths compared with the melt thickness, the formation of liquid tungsten ligaments at wave crests and their elongation by the plasma stream with splitting of the bulk of the melt, and the development of extremely long, thin threads that eventually break into liquid droplets. Ejection of these droplets in the form of fine spray can lead to significant plasma contamination and enhanced erosion of PFCs. The numerical results advance the current understanding of the physics involved in the mechanism of melt-layer breakdown and droplet generation processes. These findings may also have implications for free surface liquid metal flows considered as the first wall in the design of several types of future fusion reactors.
Growth factor stimulation induces a distinct ERα cistrome underlying breast cancer endocrine resistance
Authors: Lupien Mathieu , Meyer Clifford A. , Bailey Shannon T. , Eeckhoute Jérôme , Cook Jennifer , Westerling Thomas , Zhang Xiaoyang , Carroll Jason S. , Rhodes Daniel R. , Liu X. Shirley , Brown Myles
Published: October 2010

Estrogen receptor α (ERα) expression in breast cancer is predictive of response to endocrine therapy; however, resistance is common in ERα-positive tumors that overexpress the growth factor receptor ERBB2. Even in the absence of estrogen, ERα can be activated by growth factors, including the epidermal growth factor (EGF). EGF induces a transcriptional program distinct from estrogen; however, the mechanism of the stimulus-specific response is unknown. Here we show that the EGF-induced ERα genomic targets, its cistromes, are distinct from those induced by estrogen in a process dependent on the transcription factor AP-1. The EGF-induced ERα cistrome specifically regulates genes found overexpressed in ERBB2-positive human breast cancers. This provides a potential molecular explanation for the endocrine therapy resistance seen in ERα-positive breast cancers that overexpress ERBB2. These results suggest a central role for ERα in hormone-refractory breast tumors dependent on growth factor pathway activation and favors the development of therapeutic strategies completely antagonizing ERα, as opposed to blocking its estrogen responsiveness alone.
Phosphorylation of mammalian Sgo2 by Aurora B recruits PP2A and MCAK to centromeres
Authors: Tanno Yuji , Kitajima Tomoya S. , Honda Takashi , Ando Yasuto , Ishiguro Kei-ichiro , Watanabe Yoshinori
Published: October 2010

Shugoshin (Sgo) is a conserved centromeric protein. Mammalian Sgo1 collaborates with protein phosphatase 2A (PP2A) to protect mitotic cohesin from the prophase dissociation pathway. Although another shugoshin-like protein, Sgo2, is required for the centromeric protection of cohesion in germ cells, its precise molecular function remains largely elusive. We demonstrate that hSgo2 plays a dual role in chromosome congression and centromeric protection of cohesion in HeLa cells, while the latter function is exposed only in perturbed mitosis. These functions partly overlap with those of Aurora B, a kinase setting faithful chromosome segregation. Accordingly, we identified the phosphorylation of hSgo2 by Aurora B at the N-terminal coiled-coil region and the middle region, and showed that these phosphorylations separately promote binding of hSgo2 to PP2A and MCAK, factors required for centromeric protection and chromosome congression, respectively. Furthermore, these phosphorylations are essential for localizing PP2A and MCAK to centromeres. This mechanism seems applicable to germ cells as well. Thus, our study identifies Sgo2 as a hitherto unknown crucial cellular substrate of Aurora B in mammalian cells.
Gibberellins control fruit patterning in Arabidopsis thaliana
Authors: Arnaud Nicolas , Girin Thomas , Sorefan Karim , Fuentes Sara , Wood Thomas A. , Lawrenson Tom , Sablowski Robert , Østergaard Lars
Published: October 2010

The Arabidopsis basic helix–loop–helix (bHLH) proteins INDEHISCENT (IND) and ALCATRAZ (ALC) specify tissues required for fruit opening that have major roles in seed dispersal and plant domestication. Here, we show that synthesis of the phytohormone gibberellin is a direct and necessary target of IND, and that ALC interacts directly with DELLA repressors, which antagonize ALC function but are destabilized by gibberellin. Thus, the gibberellin/DELLA pathway has a key role in patterning the Arabidopsis fruit, and the interaction between DELLA and bHLH proteins, previously shown to connect gibberellin and light responses, is a versatile regulatory module also used in tissue patterning.
Effects of recent exposure to a conditioned stimulus on extinction of Pavlovian fear conditioning
Authors: Chan Wan Yee Macy , Leung Hiu T. , Westbrook R. Frederick , McNally Gavan P.
Published: October 2010

In six experiments we studied the effects of a single re-exposure to a conditioned stimulus (CS; “retrieval trial”) prior to extinction training (extinction-reconsolidation boundary) on the development of and recovery from fear extinction. A single retrieval trial prior to extinction training significantly augmented the renewal and reinstatement of extinguished responding. Augmentation of recovery was not observed if the retrieval and extinction training occurred in different contexts. These results contrast with those reported in earlier papers by Monfils and coworkers in rats and by Schiller and coworkers in humans. We suggest that these contrasting results could depend on the contrasting influences of either: (1) occasion-setting contextual associations vs. direct context–CS associations formed as a consequence of the retrieval trial or (2) discrimination vs. generalization between the circumstances of conditioning and extinction.
Learning under stress: The inverted-U-shape function revisited
Authors: Salehi Basira , Cordero M. Isabel , Sandi Carmen
Published: October 2010

Although the relationship between stress intensity and memory function is generally believed to follow an inverted-U-shaped curve, strikingly this phenomenon has not been demonstrated under the same experimental conditions. We investigated this phenomenon for rats’ performance in a hippocampus-dependent learning task, the radial arm water maze (RAWM). Variations in stress intensity were induced using different water temperatures (25°C, 19°C, and 16°C), which elicited increased plasma corticosterone levels. During spatial training over three consecutive days, an inverted-U shape was found, with animals trained at 19°C making fewer errors than animals trained at either higher (16°C) or lower (25°C) stress conditions. Interestingly, this function was already observed by the last trial of day 1 and maintained on the first day trial of day 2. A long-term recall probe test administered under equal temperature conditions (20°C) revealed differences in performance according to the animals’ former training conditions; i.e., platform searching for rats trained at 25°C was less accurate than for rats trained at either 16°C or 19°C. In reversal learning, groups trained at both 19°C and 25°C showed better performance than the 16°C group. We also found an interaction between anxiety and exploration traits on how individuals were affected by stressors during spatial learning. In summary, our findings confirm, for the first time, the existence of an inverted-U-shape memory function according to stressor intensity during the early learning and memory phases in a hippocampus-dependent task, and indicate the existence of individual differences related to personality-like profiles for performance at either high or low stress conditions.
In Vivo Two-Photon Calcium Imaging Using Multicell Bolus Loading
Authors: Garaschuk Olga , Konnerth Arthur
Published: October 2010

INTRODUCTION

This protocol describes an approach, multicell bolus loading (MCBL), for in vivo two-photon Ca2+ imaging of large neuronal circuits with the resolution of individual cells. The procedure was developed for in vivo imaging of the cortex, but it can be adapted easily for imaging other brain regions, including the cerebellum, olfactory bulb, or even individual spinal cord neurons. The staining technique can also be applied in brain slices of any developmental stage, from embryonic to adult. If combined with a miniature head-mounted two-photon microscope, it may allow in vivo two-photon imaging in freely moving animals.
Preparation of Early Drosophila Embryonic Fillets (Before Cuticle Deposition)
Authors: Ramachandran Preethi , Budnik Vivian
Published: October 2010

INTRODUCTION

This article describes the dissection of early Drosophila embryos to generate flat or fillet preparations. For the procedure, a modified chamber or well is designed using glass slides and sealant. Embryos are dissected by removing the chorionic (outer) membrane (performed outside the chamber) and then removing the vitelline (inner) membrane (performed inside the chamber and under saline).
Imaging Fibroblast Cells Using Atomic Force Microscopy (AFM)
Authors: Plodinec Marija , Loparic Marko , Aebi Ueli
Published: October 2010

INTRODUCTION

Atomic force microscopy (AFM) can be used to visualize the three major cytoskeletal components that contribute to the mechanical properties of the cell. These are actin microfilaments, intermediate filaments, and microtubules. In this protocol, rat embryonic fibroblasts expressing actin tagged with green fluorescent protein (GFP) are used to demonstrate this procedure.
Fixation and Preparation of Developing Tissues from Aedes aegypti: Figure 1.
Authors: Clemons Anthony , Haugen Morgan , Flannery Ellen , Kast Kristopher , Jacowski Caitlin , Severson David , Duman-Scheel Molly
Published: October 2010

INTRODUCTION

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes a method for fixation and dissection of Ae. aegypti embryos, larvae, and pupae. Tissue processed in this manner can be used subsequently for in situ hybridization detection of mRNA or immunohistochemical analysis of protein expression.
In Vitro Histone Demethylase Assay
Authors: Tsukada Yu-ichi , Nakayama Keiichi I.
Published: October 2010

INTRODUCTION

Post-translational modifications of histones play an important role in regulating chromatin dynamics and function. One of the modifications, methylation, occurs on both lysine and arginine residues, and methylation status defines the epigenetic program of a cell by determining chromatin structure and thereby regulating DNA-dependent processes such as transcription. Until recently, histone methylation was considered to be irreversible. However, the discovery of histone demethylases revealed that histone methylation is more dynamic than previously recognized. This protocol describes two different in vitro histone demethylase enzyme reactions and three different methods for measuring histone demethylase activity. The first reaction (type I) uses the Fe(II)- and α-ketoglutarate-dependent dioxygenase family of histone demethylase (represented by JmjC domain-containing histone demethylase [JHDM]); the second (type II) is for the flavin adenine dinucleotide (FAD)-dependent amine oxidase family (represented by lysine-specific demethylase 1 [LSD1]). Histone demethylase activity can then be detected by measuring the release of radiolabeled formaldehyde from 3H-labeled methylated histone substrates, by monitoring the change in methylation levels of histone substrates by immunoblotting with site-specific methylhistone antibodies, or by using mass spectrometry to detect reductions in histone peptide masses that correspond to methyl groups. These assays can be applied to a wide range of histone demethylase studies, including the measurement of histone demethylase activity in tissue and cell lysates, identification of novel histone demethylases, and screening for inhibitors of histone demethylases.
Comprehensive Analysis ofCLEPolypeptide Signaling Gene Expression and Overexpression Activity in Arabidopsis
Authors: Jun JiHyung , Fiume Elisa , Roeder Adrienne H.K. , Meng Ling , Sharma Vijay K. , Osmont Karen S. , Baker Catherine , Ha Chan Man , Meyerowitz Elliot M. , Feldman Lewis J. , Fletcher Jennifer C.
Published: October 2010

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.
A Novel Motif Essential for SNARE Interaction with the K+ Channel KC1 and Channel Gating inArabidopsis
Authors: Grefen Christopher , Chen Zhonghua , Honsbein Annegret , Donald Naomi , Hills Adrian , Blatt Michael R.
Published: October 2010

The SNARE (for soluble N-ethylmaleimide–sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K+ channel subunit KC1 and forms a tripartite complex with a second K+ channel subunit, AKT1, to control channel gating and K+ transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein–protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K+ channel gating when heterologously expressed, and that rescue of KC1-associated K+ current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE–ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.
Characterization of the W321F mutant of Mycobacterium tuberculosis catalase–peroxidase KatG
Authors: Yu Shengwei , Chouchane Salem , Magliozzo Richard S.
Published: October 2010

A single amino acid mutation (W321F) in Mycobacterium tuberculosis catalase–peroxidase (KatG) was constructed by site‐directed mutagenesis. The purified mutant enzyme was characterized using optical and electron paramagnetic resonance spectroscopy, and optical stopped‐flow spectrophotometry. Reaction of KatG(W321F) with 3‐chloroperoxybenzoic acid, peroxyacetic acid, or t‐butylhydroperoxide showed formation of an unstable intermediate assigned as Compound I (oxyferryl iron:porphyrin π‐cation radical) by similarity to wild‐type KatG, although second‐order rate constants were significantly lower in the mutant for each peroxide tested. No evidence for Compound II was detected during the spontaneous or substrate‐accelerated decay of Compound I. The binding of isoniazid, a first‐line anti‐tuberculosis pro‐drug activated by catalase–peroxidase, was noncooperative and threefold weaker in KatG(W321F) compared with wild‐type enzyme. An EPR signal assigned to a protein‐based radical tentatively assigned as tyrosyl radical in wild‐type KatG, was also observed in the mutant upon reaction of the resting enzyme with alkyl peroxide. These results show that mutation of residue W321 in KatG does not lead to a major alteration in the identity of intermediates formed in the catalytic cycle of the enzyme in the time regimes examined here, and show that this residue is not the site of stabilization of a radical as might be expected based on homology to yeast cytochrome c peroxidase. Furthermore, W321 is indicated to be important in KatG for substrate binding and subunit interactions within the dimer, providing insights into the origin of isoniazid resistance in clinically isolated KatG mutants.
Competing modes of self‐association in the regulatory domains of Bruton's tyrosine kinase: Intramolecular contact versus asymmetric homodimerization
Authors: Laederach Alain , Cradic Kendall W. , Brazin Kristine N. , Zamoon Jamillah , Fulton D. Bruce , Huang Xin‐Yun , Andreotti Amy H.
Published: October 2010

A nuclear magnetic resonance (NMR) investigation of a fragment of the nonreceptor Tec family tyrosine kinase Btk has revealed an intricate set of coupled monomer‐dimer equilibria. The Btk fragment studied contains two consecutive proline‐rich motifs followed by a single Src homology 3 (SH3) domain. We provide evidence for an asymmetric homodimer in which the amino‐terminal proline sequence of one monomer contacts the opposite SH3 binding pocket, whereas the carboxy‐terminal proline sequence of the other monomer is engaged by the second SH3 domain across the dimer interface. We show that the asymmetric homodimer structure is mimicked by a heterodimer formed in an equimolar mixture of complimentary mutants: one carrying mutations in the amino‐terminal proline stretch; the other, in the carboxy‐terminal proline motif. Moreover, a monomeric species characterized by an intramolecular complex between the amino‐terminal proline motif and the SH3 domain predominates at low concentration. Association constants were determined for each of the competing equilibria by NMR titration. The similarity of the determined Ka values reveals a delicate balance between the alternative conformational states available to Btk. Thus, changes in the local concentration of Btk itself, or co‐localization with exogenous signaling molecules that have high affinity for either proline sequence or the SH3 domain, can significantly alter species composition and regulate Btk kinase activity.
Removal of surface charge‐charge interactions from ubiquitin leaves the protein folded and very stable
Authors: Loladze Vakhtang V. , Makhatadze George I.
Published: October 2010

The contribution of solvent‐exposed charged residues to protein stability was evaluated using ubiquitin as a model protein. We combined site‐directed mutagenesis and specific chemical modifications to first replace all Arg residues with Lys, followed by carbomylation of Lys‐amino groups. Under the conditions in which all carboxylic groups are protonated (at pH 2), the chemically modified protein is folded and very stable (ΔG = 18 kJ/mol). These results indicate that surface charge–charge interactions are not an essential fundamental force for protein folding and stability.
Mechanics and dynamics of B1 domain of protein G: Role of packing and surface hydrophobic residues
Authors: Ceruso Marc A. , Amadei Andrea , Nola Alfredo Di
Published: October 2010

The structural organization of the B1 domain of streptococcal protein G (PGA) has been probed using molecular dynamics simulations, with a particular emphasis on the role of the solvent exposed Ile6 residue. In addition to the native protein (WT‐PGA), three single‐mutants (I6G‐PGA, I6F‐PGA, and I6T‐PGA), one double‐mutant (I6T, T53G‐PGA), and three isolated peptide fragments (corresponding to the helix and the two β‐hairpins) were studied in the presence of explicit water molecules. Comparative analysis of the various systems showed that the level of perturbation was directly related to the hydrophobicity and the size of the side chain of residue 6, the internal rigidity of the proteins decreasing in the order I6T‐PGA > I6G‐PGA > WT‐PGA > I6F‐PGA. The results emphasized the importance of residue 6 in controlling both the integrity of the sheet's surface and the orientation of the helix in relation to the sheet by modulation of surface/core interactions. The effects of mutations were delocalized across the structure, and glycine residues, in particular, absorbed most of the introduced strain. A qualitative structural decomposition of the native fold into elementary building‐blocks was achieved using principal component analysis and mechanical response matrices. Within this framework, internal motions of the protein were described as coordinated articulations of these structural units, mutations affecting mostly the amplitude of the motions rather than the structure/location of the building‐blocks. Analysis of the isolated peptidic fragments suggested that packing did not play a determinant role in defining the elementary building‐blocks, but that chain topology was mostly responsible.
Association of partially‐folded intermediates of staphylococcal nuclease induces structure and stability
Authors: Uversky Vladimir N. , Fink Anthony L. , Khurana Ritu , Karnoup Anton S. , Segel Daniel J. , Doniach Sebastian
Published: October 2010

Staphylococcal nuclease forms three different partially‐folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (≤0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability, and globularity of the two less‐ordered partially‐folded intermediates (A1andA2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, Fourier transform infrared spectroscopy (FTIR), and small‐angle X‐ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state.
Assignment of side‐chain conformation using adiabatic energy mapping, free energy perturbation, and molecular dynamic simulations
Authors: Frimurer Thomas M. , Olsen Ole Hvilsted , Peters Günther H. , Sørensen Morten Dahl , Led Jens JØRgen
Published: October 2010

NMR spectroscopic analysis of the C‐terminal Kunitz domain fragment (α3(VI)) from the human α3‐chain of type VI collagen has revealed that the side chain of Trp21 exists in two unequally populated conformations. The major conformation (M) is identical to the conformation observed in the X‐ray crystallographic structure, while the minor conformation (m) cannot structurally be resolved in detail by NMR due to insufficient NOE data. In the present study, we have applied: (1) rigid and adiabatic mapping, (2) free energy simulations, and (3) molecular dynamic simulations to elucidate the structure of the m conformer and to provide a possible pathway of the Trp21 side chain between the two conformers. Adiabatic energy mapping of conformations of the Trp21 side chain obtained by energy minimization identified two energy minima: One corresponding to the conformation of Trp21 observed in the X‐ray crystallographic structure and solution structure of α3(VI) (the M conformation) and the second corresponding to the m conformation predicted by NMR spectroscopy. A transition pathway between the M and m conformation is suggested. The free‐energy difference between the two conformers obtained by the thermodynamic integration method is calculated to 1.77 6 0.7 kcal/mol in favor of the M form, which is in good agreement with NMR results. Structural and dynamic properties of the major and minor conformers of the α3(VI) molecule were investigated by molecular dynamic. Essential dynamics analysis of the two resulting 800 ps trajectories reveals that when going from the M to the m conformation only small, localized changes in the protein structure are induced. However, notable differences are observed in the mobility of the binding loop (residues Thr13‐Ile18), which is more flexible in the m conformation than in the M conformation. This suggests that the reorientation of Trp21 might influence the inhibitory activity against trypsin, despite the relative large distance between the binding loop and Trp21.
The crystal structure of α‐thrombin‐hirunorm IV complex reveals a novel specificity site recognition mode
Authors: Lombardi Angela , Simone Giuseppina De , Nastri Flavia , Galdiero Stefania , Pedone Carlo , Pavone Vincenzo , Morte Rossella Della , Staiano Norma , Bolognesi Martino
Published: October 2010

The X‐ray crystal structure of the human α‐thrombin‐hirunorm IV complex has been determined at 2.5 Å resolution, and refined to an R‐factor of 0.173. The structure reveals an inhibitor binding mode distinctive of a true hirudin mimetic, which justifies the high inhibitory potency and the selectivity of hirunorm IV. This novel inhibitor, composed of 26 amino acids, interacts through the N‐terminal end with the α‐thrombin active site in a nonsubstrate mode, and binds specifically to the fibrinogen recognition exosite through the C‐terminal end. The backbone of the N‐terminal tripeptide Chg1″‐Arg2″‐2Nal3″ (Chg, cyclohexyl‐glycine; 2Nal, β‐(2‐naphthyl)‐alanine) forms a parallel β‐strand to the thrombin main‐chain segment Ser214—Gly216. The Chg1″ side chain occupies the S2 site, Arg2″ penetrates into the S1 specificity site, while the 2Nal3″ side chain occupies the aryl binding site. The Arg2″ side chain enters the S1 specificity pocket from a position quite apart from the canonical P1 site. This notwithstanding, the Arg2″ side chain establishes the typical ion pair with the carboxylate group of Asp189.
Crystal structure of the FMN‐binding domain of human cytochrome P450 reductase at 1.93 Å resolution
Authors: Zhao Qiang , Modi Sandeep , Smith Graeme , Paine Mark , Mcdonagh Paul D. , Wolf C. Roland , Tew David , Lian Lu‐Yun , Roberts Gordon C.K. , Driessen Huub P.C.
Published: October 2010

The crystal structure of the FMN‐binding domain of human NADPH‐cytochrome P450 reductase (P450R‐FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 å resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997,J Biomol NMR 10:63–75) and to the corresponding domain in the 2.6 å crystal structure of intact rat P450R (Wang M et al., 1997,Proc Nat Acad Sci USA 94:8411–8416). The crystal structure of P450R‐FMN reported here confirms the overall similarity of its α‐β‐α architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central β‐sheet. The marked similarity between P450R‐FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R‐FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P450 2D6 and to cytochromec, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochromec.
Pair potentials for protein folding: Choice of reference states and sensitivity of predicted native states to variations in the interaction schemes
Authors: Betancourt Marcos R. , Thirumalai D.
Published: October 2010

We examine the similarities and differences between two widely used knowledge‐based potentials, which are expressed as contact matrices (consisting of 210 elements) that gives a scale for interaction energies between the naturally occurring amino acid residues. These are the Miyazawa–Jernigan contact interaction matrix M and the potential matrix S derived by Skolnick J et al., 1997, Protein Sci 6:676–688. Although the correlation between the two matrices is good, there is a relatively large dispersion between the elements. We show that when Thr is chosen as a reference solvent within the Miyazawa and Jernigan scheme, the dispersion between the M and S matrices is reduced. The resulting interaction matrix B gives hydrophobicities that are in very good agreement with experiment. The small dispersion between the S and B matrices, which arises due to differing reference states, is shown to have dramatic effect on the predicted native states of lattice models of proteins. These findings and other arguments are used to suggest that for reliable predictions of protein structures, pairwise additive potentials are not sufficient. We also establish that optimized protein sequences can tolerate relatively large random errors in the pair potentials. We conjecture that three body interaction may be needed to predict the folds of proteins in a reliable manner.
Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state
Authors: Rankin Saffron E. , Watts Anthony , Roder Heinrich , Pinheiro Teresa J.T.
Published: October 2010

Unfolded apocytochrome c acquires an α‐helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt‐induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid‐inserted helical conformation proceeds via a collapsed intermediate state (IC). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme‐free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of IC through a more extended state associated with the membrane surface (IS). While IC appears to be as compact as salt‐induced compact states in solution with substantial α‐helix content, the final lipid‐inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an α‐helix content which resembles that of native cytochrome c.
Breaking the low barrier hydrogen bond in a serine protease
Authors: Kidd Richard D. , Sears Pamela , Huang Dee‐Hua , Witte Krista , Wong Chi‐Huey , Farber Gregory K.
Published: October 2010

The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water‐miscible organic co‐solvent such as N, N‐dimethylformamide (DMF). However, in 50% DMF, the kcat for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the kcat for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397 + 1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 å resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 1808 around the Cβ‐Cγ bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co‐solvents.