bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineering the nucleotide coenzyme specificity and sulfhydryl redox sensitivity of two stress-responsive aldehyde dehydrogenase isoenzymes of Arabidopsis thaliana
Authors: Stiti Naim , Adewale Isaac O. , Petersen Jan , Bartels Dorothea , Kirch Hans-Hubert
Published: February 2011

Lipid peroxidation is one of the consequences of environmental stress in plants and leads to the accumulation of highly toxic, reactive aldehydes. One of the processes to detoxify these aldehydes is their oxidation into carboxylic acids catalyzed by NAD(P)+-dependent ALDHs (aldehyde dehydrogenases). We investigated kinetic parameters of two Arabidopsis thaliana family 3 ALDHs, the cytosolic ALDH3H1 and the chloroplastic isoform ALDH3I1. Both enzymes had similar substrate specificity and oxidized saturated aliphatic aldehydes. Catalytic efficiencies improved with the increase of carbon chain length. Both enzymes were also able to oxidize α,β-unsaturated aldehydes, but not aromatic aldehydes. Activity of ALDH3H1 was NAD+-dependent, whereas ALDH3I1 was able to use NAD+ and NADP+. An unusual isoleucine residue within the coenzyme-binding cleft was responsible for the NAD+-dependence of ALDH3H1. Engineering the coenzyme-binding environment of ALDH3I1 elucidated the influence of the surrounding amino acids. Enzyme activities of both ALDHs were redox-sensitive. Inhibition was correlated with oxidation of both catalytic and non-catalytic cysteine residues in addition to homodimer formation. Dimerization and inactivation could be reversed by reducing agents. Mutant analysis showed that cysteine residues mediating homodimerization are located in the N-terminal region. Modelling of the protein structures revealed that the redox-sensitive cysteine residues are located at the surfaces of the subunits.
Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS
Authors: Chaikuad Apirat , Shafqat Naeem , Al-Mokhtar Ruby , Cameron Gus , Clarke Anthony R. , Brady R. Leo , Oppermann Udo , Frayne Jan , Yue Wyatt W.
Published: February 2011

hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN–NAD+–phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN–NAD+–glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.
Neutral sphingomyelinase 2 (nSMase2) is the primary neutral sphingomyelinase isoform activated by tumour necrosis factor-α in MCF-7 cells
Authors: Clarke Christopher J. , Cloessner Emily A. , Roddy Patrick L. , Hannun Yusuf A.
Published: February 2011

Activation of N-SMase (neutral sphingomyelinase) is an established part of the response of cytokines such as TNF (tumour necrosis factor)-α. However, it remains unclear which of the currently cloned N-SMase isoforms (nSMase1, nSMase2 and nSMase3) are responsible for this activity. In MCF-7 cells, we found that TNF-α induces late, but not early, increases in N-SMase activity, and that nSMase2 is the primary isoform activated, most likely through post-transcriptional mechanisms. Surprisingly, overexpression of tagged or untagged nSMase3 in multiple cell lines had no significant effect on in vitro N-SMase activity. Moreover, only overexpression of nSMase2, but not nSMase1 or nSMase3, had significant effects on cellular sphingolipid levels, increasing ceramide and decreasing sphingomyelin. Additionally, only siRNA (small interfering RNA) knockdown of nSMase1 significantly decreased basal in vitro N-SMase activity of MCF-7 cells, whereas nSMase2 but not nSMase3 siRNA inhibited TNF-α-induced activity. Taken together, these results identify nSMase2 as the major TNF-α-responsive N-SMase in MCF-7 cells. Moreover, the results suggest that nSMase3 may not possess in vitro N-SMase activity and does not affect cellular sphingolipid levels in the cell lines evaluated. On the other hand, nSMase1 contributes to in vitro N-SMase activity, but does not affect cellular sphingolipids much.
Nuclear envelope dynamics during plant cell division suggest common mechanisms between kingdoms
Authors: Graumann Katja , Evans David E.
Published: February 2011

Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)–GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.
Membrane-binding properties of the Factor VIII C2 domain
Authors: Novakovic Valerie A. , Cullinan David B. , Wakabayashi Hironao , Fay Philip J. , Baleja James D. , Gilbert Gary E.
Published: February 2011

Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa–Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.
A lipidomic screen of palmitate-treated MIN6 β-cells links sphingolipid metabolites with endoplasmic reticulum (ER) stress and impaired protein trafficking
Authors: Boslem Ebru , MacIntosh Gemma , Preston Amanda M. , Bartley Clarissa , Busch Anna K. , Fuller Maria , Laybutt D. Ross , Meikle Peter J. , Biden Trevor J.
Published: February 2011

Saturated fatty acids promote lipotoxic ER (endoplasmic reticulum) stress in pancreatic β-cells in association with Type 2 diabetes. To address the underlying mechanisms we employed MS in a comprehensive lipidomic screen of MIN6 β-cells treated for 48 h with palmitate. Both the overall mass and the degree of saturation of major neutral lipids and phospholipids were only modestly increased by palmitate. The mass of GlcCer (glucosylceramide) was augmented by 70% under these conditions, without any significant alteration in the amounts of either ceramide or sphingomyelin. However, flux into ceramide (measured by [3H]serine incorporation) was augmented by chronic palmitate, and inhibition of ceramide synthesis decreased both ER stress and apoptosis. ER-to-Golgi protein trafficking was also reduced by palmitate pre-treatment, but was overcome by overexpression of GlcCer synthase. This was accompanied by increased conversion of ceramide into GlcCer, and reduced ER stress and apoptosis, but no change in phospholipid desaturation. Sphingolipid alterations due to palmitate were not secondary to ER stress since they were neither reproduced by pharmacological ER stressors nor overcome using the chemical chaperone phenylbutyric acid. In conclusion, alterations in sphingolipid, rather than phospholipid, metabolism are more likely to be implicated in the defective protein trafficking and enhanced ER stress and apoptosis of lipotoxic β-cells.
Cytosolic lysine residues enhance anterograde transport and activation of the erythropoietin receptor
Authors: Yosha Liron , Ravid Orly , Ben-Califa Nathalie , Neumann Drorit
Published: February 2011

Lysine residues are key residues in many cellular processes, in part due to their ability to accept a wide variety of post-translational modifications. In the present study, we identify the EPO-R [EPO (erythropoietin) receptor] cytosolic lysine residues as enhancers of receptor function. EPO-R drives survival, proliferation and differentiation of erythroid progenitor cells via binding of its ligand EPO. We mutated the five EPO-R cytosolic lysine residues to arginine residues (5KR EPO-R), eliminating putative lysine-dependent modifications. Overexpressed 5KR EPO-R displayed impaired ubiquitination and improved stability compared with wt (wild-type) EPO-R. Unexpectedly, fusion proteins consisting of VSVGtsO45 (vesicular stomatitis virus glycoprotein temperature-sensitive folding mutant) with wt or 5KR EPO-R cytosolic domains demonstrated delayed glycan maturation kinetics upon substitution of the lysine residues. Moreover, VSVG-wt EPO-R, but not VSVG-5KR EPO-R, displayed endoplasmic reticulum-associated ubiquitination. Despite similar cell-surface EPO-binding levels of both receptors and the lack of EPO-induced ubiquitination by 5KR EPO-R, the lysine-less mutant produced weaker receptor activation and signalling than the wt receptor. We thus propose that EPO-R cytosolic lysine residues enhance receptor function, most probably through ubiquitination and/or other post-translational modifications.
Structure and mechanism of the chalcogen-detoxifying protein TehB from Escherichia coli
Authors: Choudhury Hassanul G. , Cameron Alexander D. , Iwata So , Beis Konstantinos
Published: February 2011

The oxyanion derivatives of the chalcogens tellurium and selenium are toxic to living organisms even at very low levels. Bacteria have developed mechanisms to overcome their toxicity by methylating them. The structure of TehB from Escherichia coli has been determined in the presence of the cofactor analogues SAH (S-adenosylhomocysteine) and sinefungin (a non-hydrolysable form of S-adenosyl-L-methionine) at 1.48 Å (1 Å=0.1 nm) and 1.9 Å respectively. Interestingly, our kinetic data show that TehB does not discriminate between selenium or tellurite oxyanions, making it a very powerful detoxifying protein. Analysis of the active site has identified three conserved residues that are capable of binding and orientating the metals for nucleophilic attack: His176, Arg177 and Arg184. Mutagenesis studies revealed that the H176A and R184A mutants retained most of their activity, whereas the R177A mutant had 65% of its activity abolished. Based on the structure and kinetic data we propose an SN2 nucleophilic attack reaction mechanism. These data provide the first molecular understanding of the detoxification of chalcogens by bacteria.
Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase
Authors: Zernii Evgeni Yu. , Komolov Konstantin E. , Permyakov Sergei E. , Kolpakova Tatiana , Dell'orco Daniele , Poetzsch Annika , Knyazeva Ekaterina L. , Grigoriev Ilya I. , Permyakov Eugene A. , Senin Ivan I. , Philippov Pavel P. , Koch Karl-Wilhelm
Published: February 2011

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin–kinase complex provided insight into the protein–protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation–π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.
RECQL5 is an important determinant for camptothecin tolerance in human colorectal cancer cells
Authors: Wang Xiaoqi , Lu Xincheng , Zhou Guangjin , Lou Hua , Luo Guangbin
Published: February 2011

CPTs (camptothecins) are an important class of effective anticancer agents that target type I topoisomerase in humans. Irinotecan and topotecan are currently used to treat various types of cancers and many CPT derivatives are being developed. However, these drugs are only effective in a small percentage of each type of cancer and the molecular underpinning for this individualized response to the drug has remained elusive. Thus, identification of the main determinants for cell survival in response to this unique class of drug should help to improve their clinical applications. In the present study, we examined whether RECQL5 constitutes an important determinant of CPT resistance in colon cancer cells. Specifically, RECQL5-deficient derivatives of both DDL1 and HCT116 cells, two colorectal cancer cell lines were generated by adenovirus-based somatic gene-targeting experiments and the CPT sensitivity between the RECQL5-proficient parental lines and their corresponding RECQL5-deficient derivatives were examined. We found that deletion of RECQL5 from DDL1 and HCT116 cells both resulted in a significant enhancement in CPT sensitivity under in vitro culture conditions. More importantly, xenograft tumours derived from RECQL5-deficient HCT116 cells, but not those from the parental line, could be cured by a CPT-based therapy in nude mice. Thus, the present study has identified RECQL5 as a major determinant for CPT resistance in colorectal cancer cells and a potential candidate as a biomarker for irinotecan-based treatment for colon cancer.
Ionic exclusion phase transition in neutral and weakly charged cylindrical nanopores
Authors: Buyukdagli Sahin , Manghi Manoel , Palmeri John
Published: February 2011

A field theoretic variational approach is introduced to study ion penetration into water-filled cylindrical nanopores in equilibrium with a bulk reservoir [S. Buyukdagli, M. Manghi, and J. Palmeri, Phys. Rev. Lett. 105, 158103 (2010)]. It is shown that an ion located in a neutral pore undergoes two opposing mechanisms: (i) a deformation of its surrounding ionic cloud of opposite charge, with respect to the reservoir, which increases the surface tension and tends to exclude ions from the pore, and (ii) an attractive contribution to the ion self-energy due to the increased screening with ion penetration of the repulsive image forces associated with the dielectric jump between the solvent and the pore wall. For pore radii around 1 nm and bulk concentrations lower than 0.2 mol/l, this mechanism leads to a first-order phase transition, similar to capillary “evaporation,” from an ionic-penetration state to an ionic-exclusion state. The discontinuous phase transition exists within the biological concentration range (∼0.15 mol/l) for small enough membrane dielectric constants (εm < 5). In the case of a weakly charged pore, counterion penetration exhibits a nonmonotonic behavior and is characterized by two regimes: at low reservoir concentrations or small pore radii, coions are excluded and counterions enter the pore to enforce electroneutrality; dielectric repulsion (image forces) remain strong and the counterion partition coefficient decreases with increasing reservoir concentration up to a characteristic value. For larger reservoir concentrations, image forces are screened and the partition coefficient of counterions increases with the reservoir concentration, as in the neutral pore case. Large surface charge densities (>2 × 10−3 e/nm2) suppress the discontinuous transition by reducing the energy barrier for ion penetration and shifting the critical point toward very small pore sizes and reservoir concentrations. Our variational method is also compared to a previous self-consistent approach and yields important quantitative corrections. The role of the curvature of dielectric interfaces is highlighted by comparing ionic penetration into slit and cylindrical pores. Finally, a charge regulation model is introduced in order to explain the key effect of pH on ionic exclusion and explain the origin of observed time-dependent nanopore electric conductivity fluctuations and their correlation with those of the pore surface charge.
The structure of water in the hydration shell of cations from x-ray Raman and small angle x-ray scattering measurements
Authors: Waluyo Iradwikanari , Huang Congcong , Nordlund Dennis , Bergmann Uwe , Weiss Thomas M. , Pettersson Lars G. M. , Nilsson Anders
Published: February 2011

X-ray Raman scattering (XRS) spectroscopy and small angle x-ray scattering (SAXS) are used to study water in aqueous solutions of NaCl, MgCl2, and AlCl3 with the particular aim to provide information about the structure of the hydration shells of the cations. The XRS spectra show that Na+ weakens the hydrogen bonds of water molecules in its vicinity, similar to the effect of increased temperature and pressure. Mg2+ and Al3+, on the other hand, cause the formation of short and strong hydrogen bonds between the surrounding water molecules. The SAXS data show that Mg2+ and Al3+ form tightly bound hydration shells that give a large density contrast in the scattering data. From the form factors extracted from the SAXS data, we found that Mg2+ and Al3+ have, respectively, an equivalent of one and one and a half stable hydration shells that appear as a density contrast. In addition, we estimated that the density of water in the hydration shells of Mg2+ and Al3+ is, respectively, ∼61% and ∼71% higher than in bulk water.
High precision particle mass sensing using microchannel resonators in the second vibration mode
Authors: Lee Jungchul , Bryan Andrea K. , Manalis Scott R.
Published: February 2011

An intrinsic uncertainty in particle mass sensing with the suspended microchannel resonator results from variation in a particle's position near the free end of the resonator. To circumvent this error we employ the second flexural bending mode. This mode exhibits additional frequency peaks while particles pass over the antinode, a point where the frequency shift is insensitive to the lateral position of the particle. We measure polystyrene beads with the first and second modes and confirm that the second mode sensing provides a narrower mass histogram. For 3 μm diameter beads, second mode sensing at the antinode improves the coefficient of variation in buoyant mass from 1.76% to 1.05% for population measurements and from 1.40% to 0.53% for a single trapped particle.
Wavelength selective plasmonic thermal emitter by polarization utilizing Fabry-Pérot type resonances
Authors: Chang Pei-En , Jiang Yu-Wei , Chen Hung-Hsin , Chang Yi-Tsung , Wu Yi-Ting , Tzuang Lawrence Dah-Ching , Ye Yi-Han , Lee Si-Chen
Published: February 2011

The experimental results of dispersion relations and thermal emittance spectra of a metal/insulator/metal (MIM) structure with rectangle metallic patch arrays as top layer are demonstrated. The structure exhibits wide-angle, multipeak and polarized emission characteristics caused by Fabry-Pérot type resonances of surface plasmons. Emission modes in x- and y-polarization are totally distinct, and their position depends on the width and length of the rectangular metallic patch. Therefore the designing of mode positions has two degrees of freedom and could be applied to wavelength selective light sources.
Statistical mechanics of helical wormlike chain model
Authors: Liu Ya , Pérez Toni , Li Wei , Gunton J. D. , Green Amanda
Published: February 2011

We investigate the statistical mechanics of polymers with bending and torsional elasticity described by the helical wormlike model. Noticing that the energy function is factorizable, we provide a numerical method to solve the model using a transfer matrix formulation. The tangent–tangent and binormal–binormal correlation functions have been calculated and displayed rich profiles which are sensitive to the combination of the temperature and the equilibrium torsion. Their behaviors indicate that there is no finite temperature Lifshitz point between the disordered and helical phases. The asymptotic behavior at low temperature has been investigated theoretically and the predictions fit the numerical results very well. Our analysis could be used to understand the statics of dsDNA and other chiral polymers.
Current sheath behavior and its velocity enhancement in a low energy Mather-type plasma focus device
Authors: Aghamir F. M. , Behbahani R. A.
Published: February 2011

The dynamics of the plasma sheath layer and its velocity enhancement have been studied in a low energy (4.9 kJ) Mather-type plasma focus device. Experiments were performed to study the effect of the Lorentz force variation on the current sheath expansion and movement, as well as the existence of traction between all parts of the sheath layer. Two different shape of anodes (cylindrical and step) along with an axial magnetic probe were used to investigate the effects of various experimental conditions, namely different charging voltages and gas pressures. In order to explore the upper limit of the current sheath velocity, a comparison has been made between the experimental data gathered by the probe and the Lee’s computational model. The limitations governing the enhancement of the current sheath velocity that can lead to the deterioration of a good focusing phenomenon were also investigated. The increase of the current sheath velocity due to the usage of the step anode on ion generation and hard x-ray emissions have been demonstrated by means of an ion collector and a hard x-ray detector.
Metastable decomposition and hydrogen migration of ethane dication produced in an intense femtosecond near-infrared laser field
Authors: Hoshina Kennosuke , Kawamura Haruna , Tsuge Masashi , Tamiya Minoru , Ishiguro Masaji
Published: February 2011

We investigated a formation channel of triatomic molecular hydrogen ions from ethane dication induced by irradiation of intense laser fields (800 nm, 100 fs, ∼1 × 1014 W/cm2) by using time of flight mass spectrometry. Hydrogen ion and molecular hydrogen ion (H,D)n+ (n = 1–3) ejected from ethane dications, produced by double ionization of three types of samples, CH3CH3, CD3CD3, and CH3CD3, were measured. All fragments were found to comprise components with a kinetic energy of ∼3.5 eV originating from a two-body Coulomb explosion of ethane dications. Based on the signal intensities and the anisotropy of the ejection direction with respect to the laser polarization direction, the branching ratios, H+:D+ = 66:34, H2+:HD+:D2+ = 63:6:31, and H3+:H2D+:HD2+:D3+ = 26:31:34:9 for the decomposition of C2H3D32+, were determined. The ratio of hydrogen molecules, H2:HD:D2 = 31:48:21, was also estimated from the signal intensities of the counter ion C2(H,D)42+. The similarity in the extent of H/D mixture in (H,D)3+ with that of (H,D)2 suggests that these two dissociation channels have a common precursor with the C2H42+…H2 complex structure, as proposed theoretically in the case of H3+ ejection from allene dication [A. M. Mebel and A. D. Bandrauk, J. Chem. Phys. 129, 224311 (2008)]. In contrast, the (H,D)2+ ejection path with a lower extent of H/D mixture and a large anisotropy is expected to proceed essentially via a different path with a much rapid decomposition rate. For the Coulomb explosion path of C–C bond breaking, the yield ratios of two channels, CH3CD32+→ CH3+ + CD3+ and CH2D+ + CHD2+, were 81:19 and 92:8 for the perpendicular and parallel directions, respectively. This indicates that the process occurs at a rapid rate, which is comparable to hydrogen migration through the C–C bond, resulting in smaller anisotropy for the latter channel that needs H/D exchange.
Effects of controlled ambidirectional nucleation on the heteroepitaxial growth of m-GaN on m-sapphire
Authors: Lee Hyun-Jae , Fujii K. , Goto T. , Yao T. , Chang Jiho
Published: February 2011

We found that m-plane GaN grown on m-plane sapphire nucleates in ambidirections at the initial growth stage, which seriously degrades the surface morphology and the crystallinity of m-GaN films. To avoid the ambidirectional islanding of m-plane GaN, off-cut m-plane sapphire (toward to the [112¯0] direction) substrates were introduced. When the off-angle was small as 1°–2°, the surface step determined the epitaxial orientation of m-GaN islands. Hence, an m-GaN film with a smooth surface and a low-dislocation density was obtained. However, the dislocation component of m-GaN film was increased with increasing of the off angle due to step bunches.
Negative hydrogen ion maintenance in small radius discharges: Two-dimensional modeling
Authors: Paunska Ts. , Shivarova A. , Tarnev Kh. , Tsankov Ts.
Published: February 2011

The results from a two-dimensional model of hydrogen discharges sustained in a single-chamber small radius plasma source presented in this study show that when the plasma maintenance is nonlocal, the conditions ensuring high concentration of the negative ions are formed by the behavior of the entire discharge structure and, in particular, of the fluxes in the discharge. The traditionally accepted requirements for low-electron temperature and high-electron density formulated based on the locality of the discharge behavior can no longer be employed. The obtained results show strong accumulation of negative ions in the discharge center, which results from their flux in the dc electric field, not from local balance of the ions there.
Optical response of large scale single layer graphene
Authors: Lee Chul , Kim Joo Youn , Bae Sukang , Kim Keun Soo , Hong Byung Hee , Choi E. J.
Published: February 2011

We have measured optical transmission and reflection spectra of large scale graphene grown by chemical vapor deposition technique over the extensive frequency range from far-IR to uv (4 meV–6.2 eV). Large scale graphene exhibits an excitonic absorption peak in the uv-region (ℏω=4.6 eV) and the constant interband absorption with σ1(ω)=e2/4ℏ in the IR-visible region, respectively. In the far-IR range, Drude peak is observed, and its strength ωp,2d2 indicates the induced carrier density N2d=1.95×1012 cm−2. These results are highly consistent with the theoretical prediction/experimental results of the single layer graphene. It proves that, contrary to the doubts about its quality due to the chemical growth process, the sample has single layer optical response over the entire photon energy; therefore, it can be applied to large scale devices such as terahertz-IR detector, solar cell material, and visible uv-transparent conductor.
Great thermoelectric power factor enhancement of CoSb3 through the lightest metal element filling
Authors: Zhang Jianjun , Xu Bo , Wang Li-Min , Yu Dongli , Liu Zhongyuan , He Julong , Tian Yongjun
Published: February 2011

Lithium, the lightest metal element with a small ionic radius, is successfully filled into the voids of CoSb3 by utilizing the high pressure synthesis technique. The synthesized Li0.4Co4Sb12 shows the largest thermoelectric power factor of 6000 μW m−1 K−2 among all elemental filled CoSb3 materials. This significantly enhanced thermoelectric power factor is attributed to the large carrier mobility of Li0.4Co4Sb12, 61 cm2 V−1 s−1, featuring a good electron crystal property for the Li-filled CoSb3 samples.
Investigations into Competitive Cycloaddition/Cyclization or Elimination from 1,1-Dimethyl-propargylcarbamates of Anilines
Authors: Delatouche Régis , Lesage Aurélien , Collette Floraine , Héroguez Valérie , Bertrand Philippe
Published: February 2011

The copper-catalyzed reaction of 1,1-dimethyl-O-propargyl aniline carbamates was studied and revealed the unexpected formation of oxazolidin-2-ones and alkylamines. An in-depth study of the reaction conditions showed that the formation of these products was highly dependent on the solvent, copper catalyst and aniline substituents. The reaction can be oriented towards oxazolidinones in pyridine and alkylamines in ethanol, whereas cycloaddition can be achieved in dry tetrahydrofuran.
Corrigendum to: Chemical Evidence for the Dawn of Life on Earth
Authors: Krammer Eva-Maria , Bernad Sophie , Ullmann G. Matthias , Hickman Arthur , Sebban Pierre
Published: February 2011

The dating of the dawn of life on Earth is a difficult task, requiring an accumulation of evidences from many different research fields. Here we shall summarize findings from the molecular scale (proteins) to cells and photosynthesis-related-fossils (stromatolites from the early and the late Archaean Eon), which indicate that life emerged on Earth 4.2–3.8 Ga (i.e. 4.2–3.8 × 109 years) ago. Among the data supporting this age, the isotopic and palaeontological fingerprints of photosynthesis provide some of the strongest evidence. The reason for this is that photosynthesis, carried out in particular by cyanobacteria, was responsible for massive changes to the Earth's environment, i.e. the oxygenation of the Earth's atmosphere and seawater, and the fixation of carbon from atmospheric CO2 in organic material. The possibility of a very early (>3.8 Ga ago) appearance of complex autotrophic organisms, such as cyanobacteria, is a major change in our view of life's origins.
Usefulness of winter canola (Brassica napus) race-specific resistance genes against blackleg (causal agent Leptosphaeria maculans) in southern Australian growing conditions
Authors: Light K. A. , Gororo N. N. , Salisbury P. A.
Published: February 2011

Studies on the blackleg resistance of Brassica lines containing known race-specific, Rlm resistance genes can provide information on the potential use of these genes in the genetic improvement of Australian spring canola lines. Lines of four Brassica species (winter B. napus, B. nigra, B. juncea, B. rapa) containing one or more known specific Rlm genes were assessed for seedling and adult plant survival, on infected stubble derived from crops of both polygenic and B. rapa ssp. sylvestris resistance types, to determine their potential usefulness as sources of blackleg disease resistance in diverse environments in southern Australia. Seedling and adult plant resistance of lines differed depending on the stubble type used. The seedling and adult plant blackleg resistance of several lines containing the resistance genes Rlm1, Rlm1/Rlm3, Rlm7, and Rlm10 was consistently higher than the control line, AV-Sapphire, which carries polygenic resistance. The superior performance of these lines indicates that winter B. napus and B. nigra lines have outstanding potential for improving blackleg disease resistance under Australian conditions.
Natural selection stops the evolution of male attractiveness
Authors: Hine Emma , McGuigan Katrina , Blows Mark W.
Published: February 2011

Sexual selection in natural populations acts on highly heritable traits and tends to be relatively strong, implicating sexual selection as a causal agent in many phenotypic radiations. Sexual selection appears to be ineffectual in promoting phenotypic divergence among contemporary natural populations, however, and there is little evidence from artificial selection experiments that sexual fitness can evolve. Here, we demonstrate that a multivariate male trait preferred by
Drosophila serrata
females can respond to selection and results in the maintenance of male mating success. The response to selection was associated with a gene of major effect increasing in frequency from 12 to 35% in seven generations. No further response to selection, or increase in frequency of the major gene, was observed between generations 7 and 11, indicating an evolutionary limit had been reached. Genetic analyses excluded both depletion of genetic variation and overdominance as causes of the evolutionary limit. Relaxing artificial selection resulted in the loss of 52% of the selection response after a further five generations, demonstrating that the response under artificial sexual selection was opposed by antagonistic natural selection. We conclude that male
D. serrata
sexually selected traits, and attractiveness to
D. serrata
females conferred by these traits, were held at an evolutionary limit by the lack of genetic variation that would allow an increase in sexual fitness while simultaneously maintaining nonsexual fitness. Our results suggest that sexual selection is unlikely to cause divergence among natural populations without a concomitant change in natural selection, a conclusion consistent with observational evidence from natural populations.
DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria
Authors: Shock Lisa S. , Thakkar Prashant V. , Peterson Erica J. , Moran Richard G. , Taylor Shirley M.
Published: February 2011

Mitochondrial DNA (mtDNA) has been reported to contain 5-methylcytosine (5mC) at CpG dinucleotides, as in the nuclear genome, but neither the mechanism generating mtDNA methylation nor its functional significance is known. We now report the presence of 5-hydroxymethylcytosine (5hmC) as well as 5mC in mammalian mtDNA, suggesting that previous studies underestimated the level of cytosine modification in this genome. DNA methyltransferase 1 (DNMT1) translocates to the mitochondria, driven by a mitochondrial targeting sequence located immediately upstream of the commonly accepted translational start site. This targeting sequence is conserved across mammals, and the encoded peptide directs a heterologous protein to the mitochondria. DNMT1 is the only member of the three known catalytically active DNA methyltransferases targeted to the mitochondrion. Mitochondrial DNMT1 (mtDNMT1) binds to mtDNA, proving the presence of mtDNMT1 in the mitochondrial matrix. mtDNMT1 expression is up-regulated by NRF1 and PGC1α, transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to hypoxia, and by loss of p53, a tumor suppressor known to regulate mitochondrial metabolism. Altered mtDNMT1 expression asymmetrically affects expression of transcripts from the heavy and light strands of mtDNA. Hence, mtDNMT1 appears to be responsible for mtDNA cytosine methylation, from which 5hmC is presumed to be derived, and its expression is controlled by factors that regulate mitochondrial function.
Brain-state–independent neural representation of peripheral stimulation in rat olfactory bulb
Authors: Li Anan , Gong Ling , Xu Fuqiang
Published: February 2011

It is critical for normal brains to perceive the external world precisely and accurately under ever-changing operational conditions, yet the mechanisms underlying this fundamental brain function in the sensory systems are poorly understood. To address this issue in the olfactory system, we investigated the responses of olfactory bulbs to odor stimulations under different brain states manipulated by anesthesia levels. Our results revealed that in two brain states, where the spontaneous baseline activities differed about twofold based on the local field potential (LFP) signals, the levels of neural activities reached after the same odor stimulation had no significant difference. This phenomenon was independent of anesthetics (pentobarbital or chloral hydrate), stimulating odorants (ethyl propionate, ethyl butyrate, ethyl valerate, amyl acetate, n-heptanal, or 2-heptanone), odor concentrations, and recording sites (the mitral or granular cell layers) for LFPs in three frequency bands (12–32 Hz, 33–64 Hz, and 65–90 Hz) and for multiunit activities. Furthermore, the activity patterns of the same stimulation under these two brain states were highly similar at both LFP and multiunit levels. These converging results argue the existence of mechanisms in the olfactory bulbs that ensure the delivery of peripheral olfactory information to higher olfactory centers with high fidelity under different brain states.
Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging
Authors: Guo Jia , Wang Shenliang , Dai Nan , Teo Yin Nah , Kool Eric T.
Published: February 2011

Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410–670 nm. We developed a new approach to antibody conjugation, using Huisgen–Sharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340–380 nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.
Antagonists of growth hormone-releasing hormone (GHRH) reduce prostate size in experimental benign prostatic hyperplasia
Authors: Rick Ferenc G. , Schally Andrew V. , Block Norman L. , Nadji Mehrdad , Szepeshazi Karoly , Zarandi Marta , Vidaurre Irving , Perez Roberto , Halmos Gabor , Szalontay Luca
Published: February 2011

Growth hormone-releasing hormone (GHRH), a hypothalamic polypeptide, acts as a potent autocrine/paracrine growth factor in many cancers. Benign prostatic hyperplasia (BPH) is a pathologic proliferation of prostatic glandular and stromal tissues; a variety of growth factors and inflammatory processes are inculpated in its pathogenesis. Previously we showed that potent synthetic antagonists of GHRH strongly inhibit the growth of diverse experimental human tumors including prostate cancer by suppressing various tumoral growth factors. The influence of GHRH antagonists on animal models of BPH has not been investigated. We evaluated the effects of the GHRH antagonists JMR-132 given at doses of 40 μg/d, MIA-313 at 20 μg/d, and MIA-459 at 20 μg/d in testosterone-induced BPH in Wistar rats. Reduction of prostate weights was observed after 6 wk of treatment with GHRH antagonists: a 17.8% decrease with JMR-132 treatment; a 17.0% decline with MIA-313 treatment; and a 21.4% reduction with MIA-459 treatment (P< 0.05 for all). We quantified transcript levels of genes related to growth factors, inflammatory cytokines, and signal transduction and identified significant changes in the expression of more than 80 genes (P< 0.05). Significant reductions in protein levels of IL-1β, NF-κβ/p65, and cyclooxygenase-2 (COX-2) also were observed after treatment with a GHRH antagonist. We conclude that GHRH antagonists can lower prostate weight in experimental BPH. This reduction is caused by the direct inhibitory effects of GHRH antagonists exerted through prostatic GHRH receptors. This study sheds light on the mechanism of action of GHRH antagonists in BPH and suggests that GHRH antagonists should be considered for further development as therapy for BPH.
Expression, signaling proficiency, and stimulatory function of the NKG2D lymphocyte receptor in human cancer cells
Authors: Benitez Andrea Caballero , Dai Zhenpeng , Mann Henning H. , Reeves Rebecca S. , Margineantu Daciana H. , Gooley Ted A. , Groh Veronika , Spies Thomas
Published: February 2011

The stimulatory natural killer group 2 member D (NKG2D) lymphocyte receptor and its tumor-associated ligands are important mediators in the immune surveillance of cancer. With advanced human tumors, however, persistent NKG2D ligand expression may favor tumor progression. We have found that cancer cells themselves express NKG2D in complex with the DNAX-activating protein 10 (DAP10) signaling adaptor. Triggering of NKG2D on ex vivo cancer cells or on tumor lines which express only few receptor complexes activates the oncogenic PI3K–protein kinase B (PKB/AKT)–mammalian target of rapamycin (mTOR) signaling axis and downstream effectors, the ribosomal protein S6 kinase 1 (S6K1) and the translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, as in lymphocytes, NKG2D ligand engagement stimulates phosphorylation of JNK and ERK in MAP kinase cascades. Consistent with these signaling activities, above-threshold expression of NKG2D–DAP10 in a ligand-bearing tumor line increases its bioenergetic metabolism and proliferation, thus suggesting functional similarity between this immunoreceptor and tumor growth factor receptors. This relationship is supported by significant correlations between percentages of cancer cells that are positive for surface NKG2D and criteria of tumor progression. Hence, in a conceptual twist, these results suggest that tumor co-option of NKG2D immunoreceptor expression may complement the presence of its ligands for stimulation of tumor growth.
Listeria monocytogenes
transiently alters mitochondrial dynamics during infection
Authors: Stavru Fabrizia , Bouillaud Frédéric , Sartori Anna , Ricquier Daniel , Cossart Pascale
Published: February 2011

Mitochondria are essential and highly dynamic organelles, constantly undergoing fusion and fission. We analyzed mitochondrial dynamics during infection with the human bacterial pathogen
Listeria monocytogenes
and show that this infection profoundly alters mitochondrial dynamics by causing transient mitochondrial network fragmentation. Mitochondrial fragmentation is specific to pathogenic
Listeria monocytogenes
, and it is not observed with the nonpathogenic
Listeria innocua
species or several other intracellular pathogens. Strikingly, the efficiency of
Listeria
infection is affected in cells where either mitochondrial fusion or fission has been altered by siRNA treatment, highlighting the relevance of mitochondrial dynamics for
Listeria
infection. We identified the secreted pore-forming toxin listeriolysin O as the bacterial factor mainly responsible for mitochondrial network disruption and mitochondrial function modulation. Together, our results suggest that the transient shutdown of mitochondrial function and dynamics represents a strategy used by
Listeria
at the onset of infection to interfere with cellular physiology.
Four faces of cellular senescence
Authors: Rodier Francis , Campisi Judith
Published: February 2011

Cellular senescence is an important mechanism for preventing the proliferation of potential cancer cells. Recently, however, it has become apparent that this process entails more than a simple cessation of cell growth. In addition to suppressing tumorigenesis, cellular senescence might also promote tissue repair and fuel inflammation associated with aging and cancer progression. Thus, cellular senescence might participate in four complex biological processes (tumor suppression, tumor promotion, aging, and tissue repair), some of which have apparently opposing effects. The challenge now is to understand the senescence response well enough to harness its benefits while suppressing its drawbacks.
N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2
Authors: Pratt Emily B. , Tewson Paul , Bruederle Cathrin E. , Skach William R. , Shyng Show-Ling
Published: February 2011

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (KATP) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (Po) and bursting patterns of activity observed in full-length KATP channels. However, the nature of TMD0–Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in KATP channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-KATP channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced Po, exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP2) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP2. Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP2. These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP2. Moreover, they uncover a novel mechanism of KATP channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.