bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components
Authors: Ogura Masato , Yamaki Junko , Homma Miwako K. , Homma Yoshimi
Published: July 2012

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.
Inversion technique to obtain local rotation velocity and ion temperature from line-integrated measurements for elongated tokamak plasma
Authors: Shi Y. J. , Lee S. G. , Hill K. W. , Bitter M.
Published: July 2012

An inversion technique is presented to calculate local toroidal and poloidal rotation velocity and ion temperature from line-integrated measurements of impurity lines by a matrix method. The effects of the rotation velocity on the ion temperature are analyzed in particular. An accurate inversion formula for the ion temperature is obtained. Several experimental geometries or configurations of line-integrated diagnostics in tokamaks are presented. For a plasma that is up-down symmetric, both the toroidal rotation velocity and poloidal rotation velocity can be deduced from one special line-integrated measurement.
Multi-chord fiber-coupled interferometry of supersonic plasma jets (invited)
Authors: Merritt Elizabeth C. , Lynn Alan G. , Gilmore Mark A. , Thoma Carsten , Loverich John , Hsu Scott C.
Published: July 2012

A multi-chord fiber-coupled interferometer is being used to make time-resolved density measurements of supersonic argon plasma jets on the Plasma Liner Experiment. The long coherence length of the laser (>10 m) allows signal and reference path lengths to be mismatched by many meters without signal degradation, making for a greatly simplified optical layout. Measured interferometry phase shifts are consistent with a partially ionized plasma in which both positive and negative phase shift values are observed depending on the ionization fraction. In this case, both free electrons and bound electrons in ions and neutral atoms contribute to the index of refraction. This paper illustrates how the interferometry data, aided by numerical modeling, are used to derive total jet density, jet propagation velocity (∼15–50 km/s), jet length (∼20–100 cm), and 3D expansion.
Effective dipole moment for the mode coupling instability: Mapping of self-consistent wake models
Authors: Röcker T. B. , Zhdanov S. K. , Ivlev A. V. , Lampe M. , Joyce G. , Morfill G. E.
Published: July 2012

The theory of the mode coupling instability operating in two-dimensional plasma crystals is generalized, by employing the linear plasma response formalism to describe the interparticle interactions self-consistently. In this approach, the underlying ion distribution function is calculated from first principles. Subthermal and suprathermal regimes of the ion flow are considered. A mapping procedure is proposed, which relates the self-consistent coupling coefficients to the effective dipole moment of the wake—the parameter which characterizes the mode coupling in the framework of the conventionally used Yukawa/point-wake model. The importance of the self-consistent approach is demonstrated by comparing the theoretically obtained dipole moments with the values deduced from experiments.
A combined surface stress and magneto-optical Kerr effect measurement setup for temperatures down to 30 K and in fields of up to 0.7 T
Authors: Premper J. , Sander D. , Kirschner J.
Published: July 2012

An optical 2-beam surface stress measurement and magneto-optical Kerr-effect has been combined with a liquid helium cooled cryostat. Sample temperatures down to \documentclass[12pt]{minimal}\begin{document}$\mathrm{30\;\mathrm{K}}$\end{document}30K and magnetic fields up to \documentclass[12pt]{minimal}\begin{document}$\mathrm{0.7\;\mathrm{T}}$\end{document}0.7T are achieved under UHV conditions. Low temperatures are exploited to obtain the first experimental data on the surface stress change induced by the adsorption of the noble gas Xe on Pt(111). High magnetic fields and low temperatures are used to characterize the magnetic properties of Co monolayers in longitudinal and polar Kerr geometries. The effective magnetic anisotropy is extracted from hard axis magnetization loops.
Development of a quasi-adiabatic calorimeter for the determination of the water vapor pressure curve
Authors: Mokdad S. , Georgin E. , Hermier Y. , Sparasci F. , Himbert M.
Published: July 2012

Progress in the knowledge of the water saturation curve is required to improve the accuracy of the calibrations in humidity. In order to achieve this objective, the LNE-CETIAT and the LNE-CNAM have jointly built a facility dedicated to the measurement of the saturation vapor pressure and temperature of pure water. The principle is based on a static measurement of the pressure and the temperature of pure water in a closed, temperature-controlled thermostat, conceived like a quasi-adiabatic calorimeter. A copper cell containing pure water is placed inside a temperature-controlled copper shield, which is mounted in a vacuum-tight stainless steel vessel immersed in a thermostated bath. The temperature of the cell is measured with capsule-type standard platinum resistance thermometers, calibrated with uncertainties below the millikelvin. The vapor pressure is measured by calibrated pressure sensors connected to the cell through a pressure tube whose temperature is monitored at several points. The pressure gauges are installed in a thermostatic apparatus ensuring high stability of the pressure measurement and avoiding any condensation in the tubes. Thanks to the employment of several technical solutions, the thermal contribution to the overall uncertainty budget is reduced, and the remaining major part is mainly due to pressure measurements. This paper presents a full description of this facility and the preliminary results obtained for its characterization.
Size selective spectroscopy of Se microclusters
Authors: Kooser K. , Ha D. T. , Itälä E. , Laksman J. , Urpelainen S. , Kukk E.
Published: July 2012

The electronic structure and photofragmentation in outer and inner valence regions of Sen (n ⩽ 8) clusters produced by direct vacuum evaporation have been studied with size-selective photoelectron-photoion coincidence technique by using vacuum-ultraviolet synchrotron radiation. The experimental ionization potentials of these clusters were extracted from the partial ion yield measurements. The calculations for the possible geometrical structures of the Sen microclusters have been executed. The ionization energies of the clusters have been calculated and compared with the experimental results. In addition, theoretical fragment ion appearance energies were estimated. The dissociation energies of Sen clusters were derived from the recurrent relation between the gas phase enthalpies of the formation of corresponding cationic clusters and experimental ionization energies.
Entropic stochastic resonance of a flexible polymer chain in a confined system
Authors: Zhang Zhen , Chen Hanshuang , Hou Zhonghuai
Published: July 2012

We have studied the dynamics of a flexible polymer chain in constrained dumb-bell-shape geometry subject to a periodic force and external noise along the longitudinal direction. It is found that the system exhibits a feature of entropic stochastic resonance (ESR), i.e., the temporal coherence of the polymer motion can reach a maximum level for an optimal noise intensity. We demonstrate that the occurrence of ESR is robust to the change of chain length, while the bottleneck width should be properly chosen. A gravity force in the vertical direction is not necessary for the ESR here, however, the elastic coupling between polymer beads is crucial.
About turbulence statistics in the outer part of a boundary layer developing over two-dimensional surface roughness
Authors: Krogstad Per-Åge , Efros Vladislav
Published: July 2012

The influence of surface roughness on a high Reynolds number turbulent boundary layer is investigated in the light of Townsend's wall similarity hypothesis. There is conflicting information in the literature about the effect of surface roughness on the flow structure in the outer layer. Flows over roughness consisting of spanwise rods appear to show unusually strong interaction between the wall and the wake region. It has been suggested that flows which show strong modifications in the outer layer suffer from lack of scale separation between the roughness scale and the largest scale given by the boundary layer thickness. One of the flows which have documented significant roughness effects across the entire layer is the boundary layer developing over roughness made up of spanwise aligned square bars. Here we compare one such flow taken at relatively low Reynolds number with measurements for a boundary layer which develops at much higher Reynolds number. The high Reynolds number (Reθ = 32 600) assures that the range of scale is wide, while at the same time the size of the roughness elements is sufficiently large for the flow to be fully rough. Both sets of data are compared to a smooth wall reference boundary layer and from probability distributions, the quadrant contributions to the shear stress and triple correlation profiles, we show that the outer layer changes are significantly reduced when increasing the Reynolds number and thereby the range of scales. In this case the wall perturbation of the flow is shown to be much smaller than reported in other experiments using the same geometry. Here we find that the modifications in the turbulent structure are limited to about five times the roughness scale. This is in accordance with the wall similarity hypothesis.
Er3+ interlayer energy migration as the limiting photoluminescence quenching factor in nanostructured Er3+:Y2O3 thin films
Authors: Hoang J. , Schwartz Robert N. , Wang Kang L. , Chang J. P.
Published: July 2012

We report the effects of Er3+ nanostructuring on optical properties of heterogeneously doped Er3+:Y2O3 thin films synthesized by radical enhanced atomic layer deposition. By alternating the cycle sequences of Y2O3 and Er2O3, rare earth (RE) ion concentrations were controlled from 4.8 to 11.8 at. % Er and the local Er2O3 thicknesses were varied between 0.7 to 7.6 Å. Photoluminescence (PL) was used to examine the 1535 nm (Er 4I13/2→4I15/2) emission at two excitation wavelengths, 488 nm and 976 nm. The normalized PL increased with increasing Er3+ concentrations up to 11.8 and 9.6 at. % under 488 and 976 nm excitations, respectively. The introduction of a local Er2O3 layer greater than 2.4 Å resulted in significant PL quenching, over an order of magnitude, under both excitation wavelengths. The quenching was attributed to enhanced local Er3+↔Er3+ interlayer energy migration. Compared to homogeneously doped RE systems where the RE concentration is directly related to the average RE↔RE spatial distance, increased luminescence was observed at high Er3+ concentrations in heterogeneously doped systems. These results suggest that controlling the RE proximity is key to engineering the optical properties of RE doped heterogeneous materials.
Notes on the geometric regularity criterion of 3D Navier-Stokes system
Authors: Lee Jihoon
Published: July 2012

In this note, we show that if the volume of parallelepiped which is defined by three unit vectors – direction of the velocity, direction of the vorticity, and the direction of the curl of the vorticity – is small enough, then the solution to the 3D Navier-Stokes equations is classical.
Single photon emission computed tomography-guided Cerenkov luminescence tomography
Authors: Hu Zhenhua , Chen Xueli , Liang Jimin , Qu Xiaochao , Chen Duofang , Yang Weidong , Wang Jing , Cao Feng , Tian Jie
Published: July 2012

Cerenkov luminescence tomography (CLT) has become a valuable tool for preclinical imaging because of its ability of reconstructing the three-dimensional distribution and activity of the radiopharmaceuticals. However, it is still far from a mature technology and suffers from relatively low spatial resolution due to the ill-posed inverse problem for the tomographic reconstruction. In this paper, we presented a single photon emission computed tomography (SPECT)-guided reconstruction method for CLT, in which a priori information of the permissible source region (PSR) from SPECT imaging results was incorporated to effectively reduce the ill-posedness of the inverse reconstruction problem. The performance of the method was first validated with the experimental reconstruction of an adult athymic nude mouse implanted with a Na131I radioactive source and an adult athymic nude mouse received an intravenous tail injection of Na131I. A tissue-mimic phantom based experiment was then conducted to illustrate the ability of the proposed method in resolving double sources. Compared with the traditional PSR strategy in which the PSR was determined by the surface flux distribution, the proposed method obtained much more accurate and encouraging localization and resolution results. Preliminary results showed that the proposed SPECT-guided reconstruction method was insensitive to the regularization methods and ignored the heterogeneity of tissues which can avoid the segmentation procedure of the organs.
A non-mutilating method for marking small wild mammals and reptiles
Authors: Petit Sophie , Waudby Helen P. , Walker Ashley T. , Zanker Rebecca , Rau Gina
Published: July 2012

Marking animals for identification is necessary in many aspects of wildlife research. In view of cost, practicality and ethical issues associated with many methods, we aimed to develop a suitable method for marking small wild vertebrates. We injected ultraviolet (UV) fluorescent tattoo ink subcutaneously into the tail of eight small mammal species and into the ventral area of seven lizard species. The method appeared suitable for seven mammal species and all seven lizard species, although survival after release remains to be determined. The method improved survival during handling of a sensitive rodent species compared with ear notching, which involved tighter head and chest control. Sixty-two recaptures indicated that the animals were healthy and the marks had not moved. UV-fluorescent tattoo ink is a cheap and ethical way of marking many small vertebrate species, although we recommend long-term evaluations of mark retention and the impact of tattoos on animals, including a potential increase in visibility to some predators. This marking method is likely to have applications for a wide range of wildlife.
Fullerenes: An extraterrestrial carbon carrier phase for noble gases
Authors: Becker Luann , Poreda Robert J. , Bunch Ted E.
Published: July 2012

In this work, we report on the discovery of naturally occurring fullerenes (C
60
to C
400
) in the Allende and Murchison meteorites and some sediment samples from the 65 million-year-old Cretaceous/Tertiary boundary layer (KTB). Unlike the other pure forms of carbon (diamond and graphite), fullerenes are extractable in an organic solvent (e.g., toluene or 1,2,4-trichlorobenzene). The recognition of this unique property led to the detection and isolation of the higher fullerenes in the Kratschmer/Huffmann arc evaporated graphite soot and in the carbon material in the meteorite and impact deposits. By further exploiting the unique ability of the fullerene cage structure to encapsulate and retain noble gases, we have determined that both the Allende and Murchison fullerenes and the KTB fullerenes contain trapped noble gases with ratios that can only be described as extraterrestrial in origin.
The decomposition of peroxynitrite to nitroxyl anion (NO
−
) and singlet oxygen in aqueous solution
Authors: Khan Ahsan Ullah , Kovacic Dianne , Kolbanovskiy Alexander , Desai Mehul , Frenkel Krystyna , Geacintov Nicholas E.
Published: July 2012

The mechanism of decomposition of peroxynitrite (OONO
−
) in aqueous sodium phosphate buffer solution at neutral pH was investigated. The OONO
−
was synthesized by directly reacting nitric oxide with superoxide anion at pH 13. The hypothesis was explored that OONO
−
, after protonation at pH 7.0 to HOONO, decomposes into
1
O
2
and HNO according to a spin-conserved unimolecular mechanism. Small aliquots of the concentrated alkaline OONO
−
solution were added to a buffer solution (final pH 7.0–7.2), and the formation of
1
O
2
and NO
−
in high yields was observed. The
1
O
2
generated was trapped as the transannular peroxide (DPAO
2
) of 9,10-diphenylanthracene (DPA) dissolved in carbon tetrachloride. The nitroxyl anion (NO
−
) formed from HNO (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous methemoglobin (MetHb) solution. In the presence of 25 mM sodium bicarbonate, which is known to accelerate the rate of decomposition of OONO
−
, the amount of singlet oxygen trapped was reduced by a factor of ≈2 whereas the yield of trapping of NO
−
by methemoglobin remained unaffected. Because NO
3
−
is known to be the ultimate decomposition product of OONO
−
, these results suggest that the nitrate anion is not formed by a direct isomerization of OONO
−
, but by an indirect route originating from NO
−
.
Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity
Authors: Cupp-Vickery Jill , Anderson Robert , Hatziris Zena
Published: July 2012

Several mammalian cytochrome P450 (P450) isoforms demonstrate homotropic cooperativity with a number of substrates, including steroids and polycyclic aromatic hydrocarbons. To identify structural factors contributing to steroid and polycyclic aromatic hydrocarbon binding to P450 enzymes and to determine the location of the allosteric site, we investigated interactions of the macrolide hydroxylase P450eryF from
Saccharopolyspora erythraea
with androstenedione and 9-aminophenanthrene. Spectroscopic binding assays indicate that P450eryF binds androstenedione with an affinity of 365 μM and a Hill coefficient of 1.31 ± 0.6 and coordinates with 9-aminophenanthrene with an affinity of 91 μM and a Hill coefficient of 1.38 ± 0.2. Crystals of complexes of androstenedione and 9-aminophenanthrene with P450eryF were grown and diffracted to 2.1 Å and 2.35 Å, respectively. Electron density maps indicate that for both complexes two ligand molecules are simultaneously present in the active site. The P450eryF/androstenedione model was refined to an
r
= 18.9%, and the two androstenedione molecules have similar conformations. The proximal androstenedione is positioned such that the α-face of carbon-6 is closest to the heme iron, and the second steroid molecule is positioned 5.5 Å distal in the active site. The P450eryF/9-aminophenanthrene model was refined to an
r
= 19.7% with the proximal 9-aminophenanthrene coordinated with the heme iron through the 9-amino group and the second ligand positioned ≈6 Å distal in the active site. These results establish that homotropic cooperativity in ligand binding can result from binding of two substrate molecules within the active site pocket without major conformational changes in the protein.
A method to identify cDNAs based on localization of green fluorescent protein fusion products
Authors: Misawa Kazuhide , Nosaka Tetsuya , Morita Sumiyo , Kaneko Azusa , Nakahata Tatsutoshi , Asano Shigetaka , Kitamura Toshio
Published: July 2012

We previously established a high-efficiency, retrovirus-mediated expression cloning method. Using this system, we now have developed an expression cloning method (FL-REX; fluorescence localization-based retrovirus-mediated expression cloning) in which cDNAs can be isolated based on the subcellular localization of their protein products. Complementary DNAs generated from mRNA using random hexamers were fused to the cDNA of green fluorescent protein (GFP) in the pMX retrovirus vector. The resulting cDNA-GFP fusion library was transfected into retrovirus-packaging cells, and the derived retroviruses were used to infect NIH 3T3 cells. Infected cells then were screened to identify cDNAs of interest through the subcellular localization of the GFP-fusion products. Using FL-REX, we have identified 25 cDNAs, most of which showed reasonable subcellular localization as GFP-fusion proteins, indicating that FL-REX is useful for identification of proteins that show specific intracellular localization.
Isoleucine-15 of rainbow trout carbonyl reductase-like 20β-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH) binding
Authors: Guan Guijun , Todo Takashi , Tanaka Minoru , Young Graham , Nagahama Yoshitaka
Published: July 2012

Carbonyl reductase-like 20β-hydroxysteroid dehydrogenase (CR/20β-HSD) is an enzyme that converts 17α-hydroxyprogesterone to 17α,20β-dihydroxy-4-pregnen-3-one (the maturation-inducing hormone of salmonid fish). We have previously isolated two types of CR/20β-HSD cDNAs from ovarian follicle of rainbow trout (
Oncorhynchus mykiss
). Recombinant proteins produced by expression in
Escherichia coli
in vitro
showed that one (type A) had CR and 20β-HSD activity but that the other (type B) did not. Among the three distinct residues between the protein products encoded by the two cDNAs, two residues (positions 15 and 27) are located in the N-terminal Rossmann fold, the coenzyme binding site. To investigate the structure/function relationships of CR/20β-HSDs, we generated mutants by site-directed mutagenesis at the following positions: MutA/I15T, MutB/T15I, and MutB/Q27K. Enzyme activity of wild-type A was abolished by substitution of Ile-15 by Thr (MutA/I15T). Conversely, enzyme activity was acquired by the replacement of Thr-15 with Ile in type B (MutB/T15I). MutB/T15I mutant showed properties similar to the wild-type A in every aspect tested. Mutation MutB/Q27K had only partial enzyme activity, indicating that Ile-15 plays an important role in enzyme binding of cofactor NADPH.
Unambiguous demonstration of triple-helix-directed gene modification
Authors: Barre François-Xavier , Ait-Si-Ali Slimane , Giovannangeli Carine , Luis Richard , Robin Philippe , Pritchard Linda L. , Hélène Claude , Harel-Bellan Annick
Published: July 2012

Triple-helix-forming oligonucleotides (TFOs), which can potentially
modify target genes irreversibly, represent promising tools for
antiviral therapies. However, their effectiveness on
endogenous genes has yet to be unambiguously demonstrated.
To monitor endogenous gene modification by TFOs in a yeast
model, we inactivated an auxotrophic marker gene by inserting target
sequences of interest into its coding region. The genetically
engineered yeast cells then were treated with psoralen-linked TFOs
followed by UV irradiation, thus generating highly mutagenic covalent
crosslinks at the target site whose repair could restore gene function;
the number of revertants and spectrum of mutations generated were
quantified. Results showed that a phosphoramidate TFO indeed reaches
its target sequence, forms crosslinks, and generates mutations at the
expected site via a triplex-mediated mechanism: (
i
)
under identical conditions, no mutations were generated by the same TFO
at two other loci in the target strain, nor in an isogenic control
strain carrying a modified target sequence incapable of supporting
triple-helix formation; (
ii
) for a given target
sequence, whether the triplex was formed
in vivo
on an
endogenous gene or
in vitro
on an exogenous
plasmid, the nature of the mutations generated was identical, and
consistent with the repair of a psoralen crosslink at the target site.
Although the mutation efficiency was probably too low for therapeutic
applications, our results confirm the validity of the triple-helix
approach and provide a means of evaluating the effectiveness of new
chemically modified TFOs and analogs.
Accuracy of thymine–thymine dimer bypass by
Saccharomyces cerevisiae
DNA polymerase η
Authors: Washington M. Todd , Johnson Robert E. , Prakash Satya , Prakash Louise
Published: July 2012

The
Saccharomyces cerevisiae RAD30
gene functions in error-free replication of UV-damaged DNA.
RAD30
encodes a DNA polymerase, Pol η, which inserts two adenines opposite the two thymines of a
cis
-
syn
thymine–thymine (T–T) dimer. Here we use steady-state kinetics to determine the accuracy of DNA synthesis opposite the T–T dimer. Surprisingly, the accuracy of DNA synthesis opposite the damaged DNA is nearly indistinguishable from that opposite nondamaged DNA, with frequencies of misincorporation of about 10
−2
to 10
−3
. These studies support the hypothesis that unlike most DNA polymerases, Pol η is able to tolerate distortions in DNA resulting from damage, which then enables the polymerase to utilize the intrinsic base pairing ability of the T–T dimer.
The retinoblastoma-interacting zinc-finger protein RIZ is a downstream effector of estrogen action
Authors: Abbondanza Ciro , Medici Nicola , Nigro Vincenzo , Rossi Valentina , Gallo Luigi , Piluso Giulio , Belsito Angela , Roscigno Annarita , Bontempo Paola , Puca Annibale A. , Molinari Anna Maria , Moncharmont Bruno , Puca Giovanni A.
Published: July 2012

Co-immunoprecipitation experiments in cell extract from cultured cells or target tissues indicated that estrogen receptor was complexed with the retinoblastoma binding protein RIZ in a ligand-dependent manner. Mapping of interaction sites indicated that in both proteins the same regions and motifs responsible for the interaction of transcriptional co-activator and nuclear receptors were involved. In cultured cells, estradiol induced a redistribution of RIZ protein within the nucleus and in the cytoplasm. A similar effect was produced
in vivo
, in prepuberal rat endometrium, by administration of a physiological dose of estradiol. Therefore, RIZ protein could be a specific effector of estrogen action downstream of the hormone-receptor interaction, presumably involved in proliferation control.
An approach to gene-specific transcription inhibition using oligonucleotides complementary to the template strand of the open complex
Authors: Milne Lisa , Xu Yue , Perrin David M. , Sigman David S.
Published: July 2012

The single-stranded region of DNA within the open complex of transcriptionally active genes provides a unique target for the design of gene-specific transcription inhibitors. Using the
Escherichia coli
lac
UV5 and
trp
EDCBA promoters as
in vitro
models of open complex formation, we have identified the sites inside these transcription bubbles that are accessible for hybridization by short, nuclease-resistant, nonextendible oligoribonucleotides (ORNs). Binding of ORNs inside the open complex was determined by linking the chemical nuclease bis(1,10-phenanthroline) cuprous chelate [(OP)
2
Cu
+
] to the ORN and demonstrating template-specific DNA scission. In addition, these experiments were supported by
in vitro
transcription inhibition. We find that the most effective inhibitors are 5 nt long and have sequences that are complementary to the DNA template strand in the region near the transcription start site. The ORNs bind to the DNA template strand, forming an antiparallel heteroduplex inside the open complex. In this system, RNA polymerase is essential not only to melt the duplex DNA but also to facilitate hybridization of the incoming ORN. This paradigm for gene-specific inactivation relies on the base complementarity of the ORN and the catalytic activity and sequence specificity of RNA polymerase for the site- and sequence-specific recognition and inhibition of transcriptionally active DNA.
Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers
Authors: Demmers Jeroen A. A. , Haverkamp Johan , Heck Albert J. R. , Koeppe Roger E. , Killian J. Antoinette
Published: July 2012

A method is described to study the precise positioning of transmembrane peptides in a phospholipid bilayer combining hydrogen/deuterium (H/D) exchange and nanoelectrospray ionization mass spectrometry. The method was tested by using model systems consisting of designed α-helical transmembrane peptides [acetylGW
2
(LA)
5
W
2
Aethanolamine (WALP16) and acetyl-(GA)
3
W
2
(LA)
5
W
2
(AG)
3
ethanolamine (WALP16(+10))] incorporated in large unilamellar vesicles of 1,2-dimyristoyl-
sn
-glycero-3-phosphocholine. Both peptides consist of an alternating leucine/alanine hydrophobic core sequence flanked by tryptophan residues as interfacial anchor residues. In the case of WALP16(+10), this sequence is extended at both ends by 5-aa glycine/alanine tails extending into the aqueous phase surrounding the bilayer. H/D exchange of labile hydrogens in these peptides was monitored in time after dilution of the vesicles in buffered deuterium oxide. It was found that the peptides can be measured by direct introduction of the proteoliposome suspension into the mass spectrometer. Several distinct H/D exchange rates were observed (corresponding to half-life values varying from ≤2 to ≈2 × 10
4
min). Fast exchange rates were assigned to the water-exposed tails of WALP16(+10). For both WALP16 and WALP16(+10), intermediate exchange rates were assigned to the residues close to the membrane/water interface, and the slow exchange rates to the membrane-embedded hydrophobic core. These assignments were confirmed by results from collision-induced dissociation tandem mass spectrometry experiments, which allowed analysis of exchange of individual peptide amide linkages. This proteoliposome nanoelectrospray ionization mass spectrometry technique is shown to be an extremely sensitive and powerful tool for revealing site-specific information on peptide–membrane interactions.
Evidence for secretory pathway localization of a voltage-dependent anion channel isoform
Authors: Buettner Reinhard , Papoutsoglou Georg , Scemes Eliana , Spray David C. , Dermietzel Rolf
Published: July 2012

Voltage-dependent anion channels (VDACs) are pore-forming proteins (porins) that form the major pathway for movement of adenine nucleotides through the outer mitochondrial membrane. Electrophysiological studies indicate that VDAC-like channel activity is also prevalent in the cell membranes of many mammalian cells. However, the multitopological localization of porins outside the mitochondrion has remained an extremely controversial issue. Herein, we show that usage of two alternative first exons of the murine VDAC-1 gene leads to expression of two porins differing within their N termini. One porin (plasmalemmal VDAC-1) harboring a hydrophobic leader peptide is primarily targeted through the Golgi apparatus to the cell membrane. In contrast, the second isoform lacking the N-terminal leader (mitochondrial VDAC-1) is translocated more efficiently into the outer mitochondrial membrane. Thus, our data provide unique genetic evidence in favor of a multitopological localization of a mitochondrial porin.
Signal transduction by a nondissociable heterotrimeric yeast G protein
Authors: Klein Shoshana , Reuveni Hadas , Levitzki Alexander
Published: July 2012

Many signal transduction pathways involve heterotrimeric G proteins. The accepted model for activation of heterotrimeric G proteins states that the protein dissociates to the free G
α
(GTP)-bound subunit and free G
βγ
dimer. On GTP hydrolysis, G
α
(GDP) then reassociates with G
βγ
[Gilman, A. G. (1987)
Annu. Rev. Biochem.
56, 615–649]. We reexamined this hypothesis, by using the mating G protein of the yeast
Saccharomyces cerevisiae
encoded by the genes
GPA1
,
STE4
, and
STE18
. In the absence of mating pheromone, the G
α
(Gpa1) subunit represses the mating pathway. On activation by binding of pheromone to a serpentine receptor, the G
βγ
(Ste4, Ste18) dimer transmits the signal to a mitogen-activated protein kinase cascade, leading to gene activation, arrest in the G
1
stage of the cell cycle, production of shmoos (mating projections), and cell fusion. We found that a Ste4-Gpa1 fusion protein transmitted the pheromone signal and activated the mating pathway as effectively as when Ste4 (G
β
) and Gpa1 (G
α
) were coexpressed as separate proteins. Hence, dissociation of this G protein is not required for its activation. Rather, a conformational change in the heterotrimeric complex is likely to be involved in signal transduction.
SAC3
may link nuclear protein export to cell cycle progression
Authors: Jones Amy L. , Quimby B. Booth , Hood Jennifer K. , Ferrigno Paul , Keshava Praveen H. , Silver Pamela A. , Corbett Anita H.
Published: July 2012

Selective movement of proteins between the nucleus and the cytoplasm is a regulatory mechanism exploited extensively by the eukaryotic cell. We have identified the evolutionarily conserved Sac3 protein, which was implicated previously in the regulation of mitosis [Bauer, A. & Kölling, R. (1996)
J. Cell Sci.
109, 1575–1583] as a novel mediator of nuclear protein export. We show that Sac3p is localized to the nuclear pore, where it interacts with nucleoporins. Loss of
SAC3
function results in a block in nuclear export of a nuclear export signal-containing reporter protein. Our results also demonstrate that
SAC3
interacts genetically with the nuclear protein export factors Crm1p/Xpo1p and Yrb2p. Taken together, these data indicate a link between nuclear protein export and transition through the cell cycle.
Identification and characterization of an amino acid transporter expressed differentially in liver
Authors: Gu Sumin , Roderick Hywel Llewelyn , Camacho Patricia , Jiang Jean X.
Published: July 2012

Cellular metabolic needs are fulfilled by transport of amino acids across the plasma membrane by means of specialized transporter proteins. Although many of the classical amino acid transporters have been characterized functionally, less than half of these proteins have been cloned. In this report, we identify and characterize a cDNA encoding a plasma membrane amino acid transporter. The deduced amino acid sequence is 505 residues and is highly hydrophobic with the likely predicted structure of 9 transmembrane domains, which putatively place the amino terminus in the cytoplasm and the carboxy terminus on the cell surface. Expression of the cRNA in
Xenopus laevis
oocytes revealed strong transport activities specific for histidine and glutamine. This protein is a Na
+
- and pH-dependent transporter and tolerates substitution of Na
+
by Li
+
. Furthermore, this transporter is not an obligatory exchanger because efflux occurs in the absence of influx. This transporter is expressed predominantly in the liver, although it is also present in the kidney, brain, and heart. In the liver, it is located in the plasma membrane of hepatocytes, and the strongest expression was detected in those adjacent to the central vein, gradually decreasing towards the portal tract. Because this protein displays functional similarities to the N-system amino acid transport, we have termed it mNAT, for murine N-system amino acid transporter. This is the first transporter gene identified within the N-system, one of the major amino acid transport systems in the body. The expression pattern displayed by mNAT suggests a potential role in hepatocyte physiology.
Saturation of, and competition for entry into, the apical secretory pathway
Authors: Marmorstein Alan D. , Csaky Karl G. , Baffi Judit , Lam Linda , Rahaal Firas , Rodriguez-Boulan Enrique
Published: July 2012

To investigate mechanisms of apical sorting in the secretory pathway of epithelial cells, we expressed varying amounts of the 165 amino acid isoform of vascular endothelial growth factor (VEGF
165
) and transforming growth factor β1 (TGF-β1) via replication defective adenoviruses. Apical sorting of both proteins was efficient at low expression levels but saturated or was reversed at high expression levels. High expression levels of TGF-β1 were effective at competing VEGF
165
out of the apical pathway; however, VEGF
165
did not compete out TGF-β1. Tunicamycin inhibition experiments showed that the apical polarity of VEGF
165
was independent of
N
-glycosylation. We conclude that the apical sorting of these two molecules is a saturable, signal-mediated process, involving competition for apical sorting receptors. The sorting of the two proteins does not appear to involve
N
-glycans as sorting signals, or lectin sorters. The observations are particularly relevant to gene therapy because they demonstrate that overexpression of a transgene can result in undesirable missorting of the encoded protein.
Differential requirement for p19ARF in the p53-dependent arrest induced by DNA damage, microtubule disruption, and ribonucleotide depletion
Authors: Khan Shireen Hussain , Moritsugu Janet , Wahl Geoffrey M.
Published: July 2012

p19ARF has been implicated as a key regulator of p53 stability and activation. While numerous stresses activate the p53 growth arrest pathway, those requiring p19ARF remain to be elucidated. We used p19ARF knockout mouse embryo fibroblasts to show that DNA damage and microtubule disruption require p19ARF to induce p53 responses, whereas ribonucleotide depletion and inhibition of RNA synthesis by low doses of actinomycin D do not. The data provide evidence that the arrest pathway activated by ribonucleotide depletion involves some different signal transducers than those activated by DNA damage or microtubule disruption. We also present biochemical analyses that provide insights into the mechanism by which p53 and p19ARF cooperate in normal cells to induce cell cycle arrest.
Gecko ι-crystallin: How cellular retinol-binding protein became an eye lens ultraviolet filter
Authors: Werten Paul J. L. , Röll Beate , van Aalten Daan M. F. , de Jong Wilfried W.
Published: July 2012

Eye lenses of various diurnal geckos contain up to 12% ι-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. ι-Crystallin could be either the gecko's housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko
Lygodactylus picturatus
now supports the former option. Comparison with ι-crystallin of a distantly related gecko,
Gonatodes vittatus
, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the ι-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant ι-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (≈1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. ι-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.
An archaeal genomic signature
Authors: Graham David E. , Overbeek Ross , Olsen Gary J. , Woese Carl R.
Published: July 2012

Comparisons of complete genome sequences allow the most objective and comprehensive descriptions possible of a lineage's evolution. This communication uses the completed genomes from four major euryarchaeal taxa to define a genomic signature for the Euryarchaeota and, by extension, the Archaea as a whole. The signature is defined in terms of the set of protein-encoding genes found in at least two diverse members of the euryarchaeal taxa that function uniquely within the Archaea; most signature proteins have no recognizable bacterial or eukaryal homologs. By this definition, 351 clusters of signature proteins have been identified. Functions of most proteins in this signature set are currently unknown. At least 70% of the clusters that contain proteins from all the euryarchaeal genomes also have crenarchaeal homologs. This conservative set, which appears refractory to horizontal gene transfer to the Bacteria or the Eukarya, would seem to reflect the significant innovations that were unique and fundamental to the archaeal “design fabric.” Genomic protein signature analysis methods may be extended to characterize the evolution of any phylogenetically defined lineage. The complete set of protein clusters for the archaeal genomic signature is presented as supplementary material (see the PNAS web site,
www.pnas.org
).
Adaptive diversification within a large family of recently duplicated, placentally expressed genes
Authors: Hughes Austin L. , Green Jonathan A. , Garbayo Juana M. , Roberts R. Michael
Published: July 2012

The pregnancy-associated glycoproteins (PAG) are putative peptide-binding proteins and products of a large family of genes whose expression is localized to the placental surface epithelium of artiodactyl species. We have tested the hypothesis that natural selection has favored diversification of these genes by examining patterns of nucleotide substitution in a sample of 28 closely related bovine, caprine, and ovine family members that are expressed only in trophoblast binucleate cells. Three observations were made. First, in codons encoding highly variable domains of the proteins, there was a greater accumulation of both synonymous and nonsynonymous mutations than in the more conserved regions of the genes. Second, in the variable regions, the mean number of nonsynonymous nucleotide substitutions per site was significantly greater than the mean number of synonymous substitutions per site. Third, nonsynonymous changes affecting amino acid charge occurred more frequently than expected under random substitution. This unusual pattern of nucleotide substitution implies that natural selection has acted to diversify these PAG molecules at the amino acid level, which in turn suggests that these molecules have undergone functional diversification. We estimate that the binucleate cell-expressed PAG originated 52 ± 6 million years ago, soon after the divergence of the ruminant lineage. Thus, rapid functional diversification of PAG expressed in trophoblast binucleate cells seems to have been associated with the origin of this unique placental adaptation.
Insertion site preferences of the P transposable element in
Drosophila melanogaster
Authors: Liao Guo-chun , Rehm E. Jay , Rubin Gerald M.
Published: July 2012

We determined the genomic sequence at the site of insertion in 2,266 unselected P element insertion events. Estimating physical properties of the genomic DNA at these insertion sites—such as base composition, bendability, A-philicity, protein-induced deformability, and B-DNA twist—revealed that they differ significantly from average chromosomal DNA. By examining potential hydrogen bonding sites in the major groove, we identified a 14-bp palindromic pattern centered on the 8-bp target site duplication that is generated by P element insertion. Our results suggest that the P-element transposition mechanism has a two-fold dyad symmetry and recognizes a structural feature at insertion sites, rather than a specific sequence motif.
The phytopathogenic bacteria
Erwinia carotovora
infects
Drosophila
and activates an immune response
Authors: Basset Alan , Khush Ranjiv S. , Braun Anne , Gardan Louis , Boccard Frédéric , Hoffmann Jules A. , Lemaitre Bruno
Published: July 2012

Although
Drosophila
possesses potent immune responses, little is known about the microbial pathogens that infect
Drosophila
. We have identified members of the bacterial genus
Erwinia
that induce the systemic expression of genes encoding antimicrobial peptides in
Drosophila
larvae after ingestion. These
Erwinia
strains are phytopathogens and use flies as vectors; our data suggest that these strains have also evolved mechanisms for exploiting their insect vectors as hosts.
Erwinia
infections induce an antimicrobial response in
Drosophila
larvae with a preferential expression of antibacterial versus antifungal peptide-encoding genes. Antibacterial peptide gene expression after
Erwinia
infection is reduced in two
Drosophila
mutants that have reduced numbers of hemocytes, suggesting that blood cells play a role in regulating
Drosophila
antimicrobial responses and also illustrating that this
Drosophila
–
Erwinia
interaction provides a powerful model for dissecting host–pathogen relationships.
Induction of cellular immunity by immunization with novel hybrid peptides complexed to heat shock protein 70
Authors: Moroi Yoichi , Mayhew Mark , Trcka Jiri , Hoe Mee H. , Takechi Yoshizumi , Hartl F. Ulrich , Rothman James E. , Houghton Alan N.
Published: July 2012

Heat shock proteins 70 (hsp70) derived from tissues and cells can elicit cytotoxic T lymphocyte (CTL) responses against peptides bound to hsp70. However, peptides can markedly differ in their affinity for hsp, and this potentially limits the repertoire of peptides available to induce CTL by the hsp immunization. Hybrid peptides consisting of a high-affinity ligand for the peptide-binding site of hsp70 joined to T cell epitopes by a glycine–serine–glycine linker were constructed. Immunization with hybrid peptides complexed to mouse hsp70 effectively primed specific CTL responses in mice and were more potent than T cell peptide epitopes alone with hsp70.In vivoimmunization with hsp70 and hybrid peptides led to rejection of tumors expressing antigen with greater efficacy than immunization with peptide epitope plus hsp70. Induction of CTL responses occurred independently of CD4+T cells, suggesting that immunization directly primed antigen-presenting cells to elicit CD8+cytotoxic T cell responses without T cell help. Both peptide/hsp70 complexes and mouse hsp70 alone were able to induce cultures of mouse bone marrow-derived dendritic cells (DC) to release cytokines, including DC from endotoxin-resistant C57BL/10Sc mice. Thus, hsp70/hybrid peptide complexes can activate DC for cytokine release, providing a potential adjuvant effect that could bypass T cell help.