bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron heat transport in improved confinement discharges in DIII-D
Authors: Stallard B. W. , Greenfield C. M. , Staebler G. M. , Rettig C. L. , Chu M. S. , Austin M. E. , Baker D. R. , Baylor L. R. , Burrell K. H. , DeBoo J. C. , deGrassie J. S. , Doyle E. J. , Lohr J. , McKee G. R. , Miller R. L. , Peebles W. A. , Petty C. C. , Pinsker R. I. , Rice B. W. , Rhodes T. L. , Waltz R. E. , Zeng L. ,   The DIII-D Team
Published: July 2002

In DIII-D [J. L. Luxon and L. G. Davis, Fusion Technol. 8, 441 (1985)] tokamak plasmas with an internal transport barrier (ITB), the comparison of gyrokinetic linear stability (GKS) predictions with experiments in both low and strong negative magnetic shear plasmas provide improved understanding for electron thermal transport within the plasma. Within a limited region just inside the ITB, the electron temperature gradient (ETG) modes appear to control the electron temperature gradient and, consequently, the electron thermal transport. The increase in the electron temperaturegradient with more strongly negative magnetic shear is consistent with the increase in the ETG mode marginal gradient. Closer to the magnetic axis the Te profile flattens and the ETG modes are predicted to be stable. With additional core electron heating, FIR scattering measurements near the axis show the presence of high k fluctuations (12 cm−1), rotating in the electron diamagnetic drift direction. This turbulence could impact electron transport and possibly also ion transport. Thermal diffusivities for electrons, and to a lesser degree ions, increase. The ETG mode can exist at this wave number, but it is computed to be robustly stable near the axis. Consequently, in the plasmas we have examined, calculations of drift wave linear stability do not explain the observed transport near the axis in plasmas with or without additional electron heating, and there are probably other processes controling transport in this region.
Dust-plasma sheath: A new dissipative self-organized structure
Authors: Tsytovich V. N. , Vladimirov S. V. , Benkadda S.
Published: July 2002

It is shown that the presence of dust near the wall of a low-temperature plasma leads to formation of a dissipative structure confining dust and plasma particles and creating the specific dust-plasma layer. This equilibrium structure is formed self-consistently with the fields of dust particles. It is demonstrated that all properties of the dust-plasma sheath depend only on one parameter—the March number of the ion flow, which have allowed zones and can be less than unity, in contrast to the features of the plasma sheath without influence of dust.
Quantum surface wave on a thin plasma layer
Authors: Shokri B. , Rukhadze A. A.
Published: July 2002

The results of theoretical analysis of the oscillation spectrum of a thin plasma layer placed in a very strong quantizing magnetic field are presented. It is shown that in the ultra-quantum limit and when the wave velocity is much larger than the ion and electron thermal velocity, quantum effects appear only if Z≠1, where Z=|ei/e| is the ion–electron charge ratio. In the two-component plasma with Z≠1 surface quantum waves are unstable, whereas in the one component plasma they are damping. At the same time, in the classical limit of magneto-confined plasma, surface waves excitation takes place and their frequency spectrum is placed in the microwave frequency range and, therefore, can be used for measuring the strength of very strong magnetic fields.
Two-dimensional particle-in-cell simulation for magnetized transport of ultra-high relativistic currents in plasma
Authors: Honda M. , Meyer-ter-Vehn J. , Pukhov A.
Published: July 2002

Nonlinear channel dynamics and magnetized transport of relativistic electron currents in plasma have been investigated, using transverse two-dimensional particle-in-cell simulations allowing for movable ions and fully relativistic binary collisions. Current filaments self-organize in coaxial structures where the relativistic beam in the center is surrounded by magnetized vacuum and a thin return current sheath outside. The current sheath explodes radially. The filament as a whole is current-neutral with almost vanishing magnetic field at the outside. Ion dynamics play an important role, leading to enhanced self-pinching of the filament cores. Collisional effects become significant in the slowly moving return currents. It is shown that electron currents of 100–1000 MA can be transported through dense plasma, but only through a large number of current filaments, each carrying about one Alfvén current. This aspect is essential for relativistic electron transport in fast ignition of targets for inertial confinement fusion (ICF).
Mechanism of dust-acoustic instability in a direct current glow discharge plasma
Authors: Fortov V. E. , Khrapak A. G. , Khrapak S. A. , Molotkov V. I. , Nefedov A. P. , Petrov O. F. , Torchinsky V. M.
Published: July 2002

An observation of low frequency waves spontaneously excited in a dc glow discharge dusty plasma is reported. To analyze possible reasons for the instability observed, a linear dispersion relation which takes into account collisions with neutrals, dust grain charge variations, ion drift, and forces acting on dust particles is derived. Numerical analysis of the dispersion relation shows that the observed instability is the result of dust charge variations in the presence of external charge-dependent forces together with the ion drift effect.
Impurity-induced turbulence suppression and reduced transport in the DIII-D tokamak
Authors: McKee G. R. , Murakami M. , Boedo J. A. , Brooks N. H. , Burrell K. H. , Ernst D. R. , Fonck R. J. , Jackson G. L. , Jakubowski M. , La Haye R. J. , Messiaen A. M. , Ongena J. , Rettig C. L. , Rice B. W. , Rost C. , Staebler G. M. , Sydora R. D. , Thomas D. M. , Unterberg B. , Wade M. R. , West W. P.
Published: July 2002

Long wavelength turbulence as well as heat and momentum transport are significantly reduced in the DIII-D tokamak [Plasma Physics and Controlled Nuclear Fusion Research (International Atomic Energy Agency, Vienna, 1987), Vol. I, p. 159] as a result of neon seeding of a low confinement mode negative central shear discharge. Correspondingly, the energy confinement time increases by up to 80%. Fully saturated turbulence measurements near ρ=0.7 (ρ=r/a) in the wave number range 0.1⩽k⊥ρs⩽0.6, obtained with beam emission spectroscopy, exhibit a significant reduction of fluctuation power after neon injection. Fluctuation measurements obtained with far infrared scattering also show a reduction of turbulence in the core, while the Langmuir probe array measures reduced particle flux in the edge and scrape-off layer. Gyrokinetic linear stability simulations of these plasmas are qualitatively consistent, showing a reduction in the growth rate of ion temperature gradient driven modes for 0<k⊥ρs⩽1.4, and nonlinear gyrokinetic simulations show a reduced saturated density fluctuation amplitude. The measured ωE×B shearing rate increased at ρ=0.7, suggesting that impurity-induced growth rate reduction is acting synergistically with ωE×B shear to decrease turbulence and reduce anomalous transport.
Radial discontinuities in tokamak magnetohydrodynamic equilibria with poloidal flow
Authors: Betti R. , Freidberg J. P.
Published: July 2002

It is shown that transonic poloidal flow leads to ideal magnetohydrodynamic tokamak equilibria with radial discontinuities in the density, pressure, and flow velocity profiles. Transonic profiles are defined as having flow velocities ranging from subsonic to supersonic with respect to the poloidal sound speed (csBp/B). The jump of the equilibrium quantities occurs approximately at the sonic surface and its magnitude is of order ε1/2 (ε is the inverse aspect ratio). Because of the large velocity shear at the sonic surface, transonic profiles may improve energy confinement as suggested by current understanding of tokamak plasma turbulence suppression.
Highly efficient neodymium:yttrium aluminum garnet laser end pumped by a semiconductor laser array
Authors: Sipes D. L.
Published: July 2002

In recent experiments, 80-mW cw power in a single mode has been achieved from a neodymium: yttrium aluminum garnet (Nd:YAG) laser with only 1 W of electrical power input to a single semiconductor laser array pump. This corresponds to an overall efficiency of 8%, the highest reported cw efficiency for a Nd:YAG laser. A tightly focused semiconductor laser end pump configuration is used to achieve high pumping intensities (on the order of 1 kW/cm2), which in turn causes the photon to photon conversion efficiency to approach the quantum efficiency (76% for Nd:YAG at 1.06 μm pumped at 0.810 μm). This is achieved despite the dual-lobed nature of the pump. Through the use of simple beam-combining schemes (e.g., polarization coupling and multireflection point pumping), output powers over 1 W and overall electrical to optical efficiencies as high as 10% are expected.
Collection mode near-field scanning optical microscopy
Authors: Betzig E. , Isaacson M. , Lewis A.
Published: July 2002

Super-resolution imaging at optical wavelengths has been achieved with collection mode near-field scanning optical microscopy. Reproducible images of 0.25-μm aluminum lines separated by 0.25 μm have been generated with a peak edge sharpness of 0.07 μm. Images taken with differing probe sizes and at various heights demonstrate that the smallest resolvable features are roughly determined by the greater of the aperture size and the aperture to sample separation.
Phase locking of Josephson junction arrays
Authors: Hadley P. , Beasley M. R. , Wiesenfeld K.
Published: July 2002

We report the results of a stability analysis of coherent oscillations in series arrays of Josephson junctions with a matched resistive load. We find that arbitrarily large, dc biased arrays of Josephson junctions will phase lock most strongly when the capacitance parameter βc ≊1, and the bias current is about twice the critical current of the individual junctions.
Combining multiple structure and sequence alignments to improve sequence detection and alignment: Application to the SH2 domains of Janus kinases
Authors: Al-Lazikani Bissan , Sheinerman Felix B. , Honig Barry
Published: July 2002

In this paper, an approach is described that combines
multiple structure alignments and multiple sequence alignments
to generate sequence profiles for protein families. First, multiple
sequence alignments are generated from sequences that are closely
related to each sequence of known three-dimensional structure. These
alignments then are merged through a multiple structure alignment of
family members of known structure. The merged alignment is used to
generate a Hidden Markov Model for the family in question. The Hidden
Markov Model can be used to search for new family members or to
improve alignments for distantly related family members that already
have been identified. Application of a profile generated for SH2
domains indicates that the Janus family of nonreceptor protein tyrosine
kinases contains SH2 domains. This conclusion is strongly supported by
the results of secondary structure-prediction programs, threading
calculations, and the analysis of comparative models generated for
these domains. One of the Janus kinases, human TYK2, has an SH2 domain
that contains a histidine instead of the conserved arginine at the key
phosphotyrosine-binding position, βB5. Calculations of the
pK
a
values of the βB5 arginines in a number of SH2
domains and of the βB5 histidine in a homology model of TYK2 suggest
that this histidine is likely to be neutral around pH 7, thus
indicating that it may have lost the ability to bind phosphotyrosine.
If this indeed is the case, TYK2 may contain a domain with an SH2 fold
that has a modified binding specificity.
Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome
Authors: Funakoshi Minoru , Sasaki Toru , Nishimoto Takeharu , Kobayashi Hideki
Published: July 2002

Dsk2p from
Saccharomyces cerevisiae
belongs to the class of proteins that contain a ubiquitin-like (UbL) domain at the N terminus together with a ubiquitin-associated (UBA) domain at the C terminus. We show here that the C-terminal UBA domain of Dsk2p binds to K48-linked polyubiquitin chains, and the N-terminal UbL domain of Dsk2p interacts with the proteasome. Overexpression of Dsk2p caused the accumulation of large amounts of polyubiquitin, and extragenic suppressors of the Dsk2p-mediated lethality proved to be temperature-sensitive mutations in two proteasome subunits,
rpn1
and
pre2.
K48-linked ubiquitin-dependent degradation was impaired by disruption of the
DSK2
gene. These results indicate that Dsk2p is K48-linked polyubiquitin-binding protein and also interacts with the proteasome. We discuss a possible role of adaptor function of Dsk2p via its UbL and UBA domains in the ubiquitin-proteasome pathway.
Quaternary structure of human fatty acid synthase by electron cryomicroscopy
Authors: Brink Jacob , Ludtke Steven J. , Yang Chao-Yuh , Gu Zei-Wei , Wakil Salih J. , Chiu Wah
Published: July 2002

We present the first three-dimensional reconstruction of
human fatty acid synthase obtained by electron cryomicroscopy and
single-particle image processing. The structure shows that the
synthase is composed of two monomers, arranged in an antiparallel
orientation, which is consistent with biochemical data. The
monomers are connected to each other at their middle by a bridge of
density, a site proposed to be the combination of the interdomain
regions of the two monomers. Each monomer subunit appears to be
subdivided into three structural domains. With this
reconstruction of the synthase, we propose a location for the
enzyme's two fatty acid synthesis sites.
Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture
Authors: Contreras Jorge E. , Sánchez Helmut A. , Eugenín Eliseo A. , Speidel Dina , Theis Martin , Willecke Klaus , Bukauskas Feliksas F. , Bennett Michael V. L. , Sáez Juan C.
Published: July 2002

Rat cortical astrocytes in pure culture are functionally coupled to
neighboring cells via connexin (Cx) 43 gap junctions under
ordinary conditions. Small fluorescent molecules such as Lucifer yellow
(LY) pass between cell interiors via gap junctions, but do not enter
the cells when externally applied. Subjecting rat and mouse cortical
astrocytes to “chemical ischemia” by inhibition of glycolytic and
oxidative metabolism induced permeabilization of cells to Lucifer
yellow and ethidium bromide before loss of membrane integrity
determined by dextran uptake and lactate dehydrogenase release. The gap
junction blockers octanol and 18α-glycyrrhetinic acid markedly
reduced dye uptake, suggesting that uptake was mediated by opening of
unapposed hemichannels. Extracellular La
3+
also reduced dye
uptake and delayed cell death. The purinergic blocker, oxidized ATP,
was ineffective. Astrocytes isolated from mice with targeted deletion
of the Cx43 coding DNA exhibited greatly reduced dye coupling and
ischemia-induced dye uptake, evidence that dye uptake is mediated by
Cx43 hemichannels. Dye coupling was reduced but not blocked by
metabolic inhibition. Blockade of lipoxygenases or treatment
with free radical scavengers reduced dye uptake by rat astrocytes,
suggesting a role for arachidonic acid byproducts in hemichannel
opening. Furthermore, permeabilization was accompanied by reduction in
ATP levels and dephosphorylation of Cx43. Although hemichannel opening
would tend to collapse electrochemical and metabolic gradients across
the plasma membrane of dying cells, healthy cells might rescue dying
cells by transfer of ions and essential metabolites via Cx43 gap
junctions. Alternatively, dying astrocytes might compromise the health
of neighboring cells via Cx43 gap junctions, thereby promoting the
propagation of cell death.
Differential aggregation of the
Trembler
and
Trembler J
mutants of peripheral myelin protein 22
Authors: Tobler Andreas R. , Liu Ning , Mueller Lukas , Shooter Eric M.
Published: July 2002

Mutations in the gene encoding the peripheral myelin
protein 22 (PMP22), a tetraspan protein in compact peripheral myelin,
are one of the causes of inherited demyelinating peripheral neuropathy.
Most
PMP22
mutations alter the trafficking of the PMP22
protein in Schwann cells, and this different trafficking has been
proposed as the underlying mechanism of the disease. To explore this
problem further, we compared the aggregation of wild-type Pmp22 with
those of the two
Pmp22
mutations found in
Trembler
(
Tr
) and
Trembler
J
(
TrJ
) mice. All three Pmp22s can be
crosslinked readily as homodimers in transfected cells. Wild-type Pmp22
also forms heterodimers with Tr and TrJ Pmp22, and these heterodimers
traffic with their respective mutant Pmp22 homodimers. All three Pmp22s
form complexes larger than dimers with Tr Pmp22 especially prone to
aggregate into high molecular weight complexes. Despite the differences
in aggregation of Tr and TrJ Pmp22, these two mutant Pmp22s sequester
the same amount of wild-type Pmp22 in heterodimers and heterooligomers.
Thus, the differences in the phenotypes of
Tr
and
TrJ
mice may depend more on the ability of the mutant
protein to aggregate than on the dominant-negative effect of the mutant
Pmp22 on wild-type Pmp22 trafficking.
Determination of causal connectivities of species in reaction networks
Authors: Vance William , Arkin Adam , Ross John
Published: July 2002

We present an approach to the determination of causal connectivities and part of the kinetics of complex reaction systems. Our approach is based on analytical and computational methods for studying the effects of a pulse change of concentration of a chemical species in a reaction network, either at equilibrium or in a nonequilibrium stationary state. Such disturbances generally propagate through a few species, depending on the values of the kinetic coefficients, before being broadened and dissipated. This short range gives a local probe of the kinetics and connectivity of the reaction network. The range of propagation also indicates species to perturb in further experiments. From piecing together these local connectivities, the global structure of the network can be constructed. The experimental design allows deduction of both reaction orders and rate constants in many cases. An example of the usefulness of the approach is illustrated on a model of a part of glycolysis.
Mg
2+
-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules
Authors: Kim Harold D. , Nienhaus G. Ulrich , Ha Taekjip , Orr Jeffrey W. , Williamson James R. , Chu Steven
Published: July 2002

Fluorescence correlation spectroscopy (FCS) of fluorescence resonant energy transfer (FRET) on immobilized individual fluorophores was used to study the Mg
2+
-facilitated conformational change of an RNA three-helix junction, a structural element that initiates the folding of the 30S ribosomal subunit. Transitions of the RNA junction between open and folded conformations resulted in fluctuations in fluorescence by FRET. Fluorescence fluctuations occurring between two FRET states on the millisecond time scale were found to be dependent on Mg
2+
and Na
+
concentrations. Correlation functions of the fluctuations were used to determine transition rates between the two conformations as a function of Mg
2+
or Na
+
concentration. Both the opening and folding rates were found to vary with changing salt conditions. Assuming specific binding of divalent ions to RNA, the Mg
2+
dependence of the observed rates cannot be explained by conformational change induced by Mg
2+
binding/unbinding, but is consistent with a model in which the intrinsic conformational change of the RNA junction is altered by uptake of Mg
2+
ion(s). This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.
Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling
Authors: Lafont Frank , Simons Kai
Published: July 2002

The high affinity receptor for IgE, FcɛRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcɛRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcɛRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcɛRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcɛRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcɛRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcɛRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.
Neutrophils lacking phosphoinositide 3-kinase γ show loss of directionality duringN-formyl-Met-Leu-Phe-induced chemotaxis
Authors: Hannigan Michael , Zhan Lijun , Li Zhong , Ai Youxi , Wu Dianqing , Huang Chi-Kuang
Published: July 2002

Confocal imaging and time-lapsed videomicroscopy were used to study the directionality, motility, rate of cell movement, and morphologies of phosphoinositide 3-kinase γ (PI3K)γ−/−neutrophils undergoing chemotaxis in Zigmond chambers containingN-formyl-Met-Leu-Phe gradients. Most of thePI3Kγ−/−neutrophils failed to translocate up the cheomotactic gradient. A partial reduction in cell motility and abnormal morphologies were also observed. In the wild-type neutrophils, the pleckstrin homology domain-containing protein kinase B (AKT) and F-actin colocalize to the leading edge of polarized neutrophils oriented toward the gradient, which was not observed inPI3Kγ−/−neutrophils. InPI3Kγ−/−neutrophils, AKT staining consistently failed to perfectly overlap with the F-actin. This failure was observed as an F-actin-filled region of 2.3 ± 0.5 μm between AKT and the cell membrane. These data suggest that PI3Kγ regulates neutrophil chemotaxis primarily by controlling the direction of cell migration and the intracellular colocalization of AKT and F-actin to the leading edge.
Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-γ
Authors: DaFonseca Christopher J. , Shu Fong , Zhang J. Jillian
Published: July 2002

In response to IFN-γ, the latent cytoplasmic Stat1 (signal transducer and activator of transcription) proteins translocate into the nucleus and activate transcription. We showed previously that Stat1 recruits a group of nuclear proteins, among them MCM5 (minichromosome maintenance) and MCM3, for transcription activation. MCM5 directly interacts with the transcription activation domain (TAD) of Stat1 and enhances Stat1-mediated transcription activation. In this report, we identified two specific residues (R732, K734) in MCM5 that are required for the direct interaction between Stat1 and MCM5 bothin vitroandin vivo. MCM5 containing mutations of R732/K734 did not enhance Stat1-mediated transcription activation in response to IFN-γ. In addition, it also failed to form complexes with other MCM proteinsin vivo, suggesting that these two residues may be important for an interaction domain in MCM5. Furthermore, MCM5 bearing mutations in its ATPase and helicase domains did not enhance Stat1 activity.In vitrobinding assays indicate that MCM3 does not interact directly with Stat1, suggesting that the presence of MCM3 in the group of Stat1TAD-interacting proteins is due to the association of MCM3 with MCM5. Finally, gel filtration analyses of nuclear extracts from INF-γ-treated cells demonstrate that there is a MCM5/3 subcomplex coeluting with Stat1. Together, these results strongly suggest that Stat1 recruits a MCM5/3 subcomplex through direct interaction with MCM5 in the process of IFN-γ-induced gene activation.
Dynamics of self assembly of magnetized disks rotating at the liquid–air interface
Authors: Grzybowski Bartosz A. , Stone Howard A. , Whitesides George M.
Published: July 2002

This paper is a theoretical study of dynamic self assembly in a system of millimeter-sized magnetized disks floating at a liquid–air interface and spinning under the influence of a rotating magnetic field. Equations of motions are derived that account for the hydrodynamic and magnetic forces acting in the system. Numerical integration of these equations predicts formation of ordered structures of spinning disks; the simulated structures reproduce the patterns observed experimentally.
Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia
Authors: Delgado Julio C. , Tsai Eunice Y. , Thim Sok , Baena Andres , Boussiotis Vassiliki A. , Reynes Jean-Marc , Sath Sun , Grosjean Pierre , Yunis Edmond J. , Goldfeld Anne E.
Published: July 2002

Purified protein derivative (PPD) skin testing is used to identify persons infected withMycobacterium tuberculosis(Mtb) and to assess cell-mediated immune responses to Mtb. However, lack of skin induration to intradermal injection of PPD or PPD anergy is observed in a subset of patients with active tuberculosis (TB). To investigate the sensitivity and persistence of PPD reactivity and itsin vitrocorrelates during active TB disease and after successful chemotherapy, we evaluated the distribution of skin size induration after intradermal injection of PPD among 364 pulmonary TB patients in Cambodia. A subset of 25 pulmonary TB patients who had a positive skin reaction to mumps and/or candida antigens showed persistent anergy to PPD after successful completion of TB therapy. Strikingly,in vitrostimulation of T cells from persistently anergic TB patients with mumps but not PPD resulted in T cell proliferation, and lower levels of IL-2 and IFN-γ and higher levels of IL-10 were detected in PPD-stimulated cellular cultures from PPD-anergic as compared with PPD-reactive pulmonary TB patients. These results show that anergy to PPD is antigen-specific and persistent in a subset of immunocompetent pulmonary TB patients and is characterized by antigen-specific impaired T cell proliferative responses and a distinct pattern of cytokine production including reduced levels of IL-2.
Crystallographic snapshots along a protein-induced DNA-bending pathway
Authors: Horton Nancy C. , Perona John J.
Published: July 2002

Two new high-resolution cocrystal structures of
Eco
RV endonuclease bound to DNA show that a large variation in DNA-bending angles is sampled in the ground state binary complex. Together with previous structures, these data reveal a contiguous series of protein conformational states delineating a specific trajectory for the induced-fit pathway. Rotation of the DNA-binding domains, together with movements of two symmetry-related helices binding in the minor groove, causes base unstacking at a key base-pair step and propagates structural changes that assemble the active sites. These structures suggest a complex mechanism for DNA bending that depends on forces generated by interacting protein segments, and on selective neutralization of phosphate charges along the inner face of the bent double helix.
Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation
Authors: Lu Shelly C. , Alvarez Luis , Huang Zong-Zhi , Chen Lixin , An Wei , Corrales Fernando J. , Avila Matías A. , Kanel Gary , Mato José M.
Published: July 2002

Liver-specific and nonliver-specific methionine
adenosyltransferases (MATs) are products of two genes,
MAT1A
and
MAT2A
, respectively, that
catalyze the formation of
S
-adenosylmethionine (AdoMet),
the principal biological methyl donor. Mature liver expresses
MAT1A
, whereas
MAT2A
is expressed in
extrahepatic tissues and is induced during liver growth and
dedifferentiation. To examine the influence of
MAT1A
on
hepatic growth, we studied the effects of a targeted disruption of the
murine
MAT1A
gene.
MAT1A
mRNA and protein
levels were absent in homozygous knockout mice. At 3 months, plasma
methionine level increased 776% in knockouts. Hepatic AdoMet and
glutathione levels were reduced by 74 and 40%, respectively, whereas
S
-adenosylhomocysteine, methylthioadenosine, and global
DNA methylation were unchanged. The body weight of 3-month-old knockout
mice was unchanged from wild-type littermates, but the liver weight was
increased 40%. The Affymetrix
genechip
system and Northern
and Western blot analyses were used to analyze differential expression
of genes. The expression of many acute phase-response and inflammatory
markers, including orosomucoid, amyloid, metallothionein, Fas
antigen, and growth-related genes, including early growth response 1
and proliferating cell nuclear antigen, is increased in the knockout
animal. At 3 months, knockout mice are more susceptible to
choline-deficient diet-induced fatty liver. At 8 months, knockout mice
developed spontaneous macrovesicular steatosis and predominantly
periportal mononuclear cell infiltration. Thus, absence of
MAT1A
resulted in a liver that is more susceptible to
injury, expresses markers of an acute phase response, and displays
increased proliferation.
Cooperative enhancement of specificity in a lattice of T cell receptors
Authors: Chan Cliburn , George Andrew J. T. , Stark Jaroslav
Published: July 2002

Two of the most important models to account for the specificity and sensitivity of the T cell receptor (TCR) are the kinetic proofreading and serial ligation models. However, although kinetic proofreading provides a means for individual TCRs to measure accurately the length of time they are engaged and signal appropriately, the stochastic nature of ligand dissociation means the kinetic proofreading model implies that at high concentrations the response of the cell will be relatively nonspecific. Recent ligand experiments have revealed the phenomenon of both negative and positive crosstalk among neighboring TCRs. By using a Monte Carlo simulation of a lattice of TCRs, we integrate receptor crosstalk with the kinetic proofreading and serial ligation models and discover that receptor cooperativity can enhance T cell specificity significantly at a very modest cost to the sensitivity of the response.
Proteinase inhibitors I and II from potatoes block UVB-induced AP-1 activity by regulating the AP-1 protein compositional patterns in JB6 cells
Authors: Liu Guangming , Chen Nanyue , Kaji Akira , Bode Ann M. , Ryan Clarence A. , Dong Zigang
Published: July 2002

Proteinase inhibitor I (Inh I) and proteinase inhibitor II (Inh II)
from potato tubers are effective proteinase inhibitors of
chymotrypsin and trypsin. Inh I and Inh II were shown to
suppress irradiation-induced transformation in mouse embryo fibroblasts
suggesting that they possess anticarcinogenic characteristics. We have
previously demonstrated that Inh I and Inh II could effectively block
UV irradiation-induced activation of transcription activator protein 1
(AP-1) in mouse JB6 epidermal cells, which mechanistically may explain
their anticarcinogenic actions. In the present study, we investigated
the effects of Inh I and Inh II on the expression and composition
pattern of the AP-1 complex following stimulation by UV B (UVB)
irradiation in the JB6 model. We found that Inh I and Inh II
specifically inhibited UVB-induced AP-1, but not NFκB, activity in
JB6 cells. Both Inh I and Inh II up-regulated AP-1 constituent
proteins, JunD and Fra-2, and suppressed c-Jun and c-Fos expression and
composition in bound AP-1 in response to UVB stimulation. This
regulation of the AP-1 protein compositional pattern in response to Inh
I or Inh II may be critical for the inhibition of UVB-induced AP-1
activity by these agents found in potatoes.
Lifetimes of intermediates in the β-sheet to α-helix transition of β-lactoglobulin by using a diffusional IR mixer
Authors: Kauffmann Ekkehard , Darnton Nicholas C. , Austin Robert H. , Batt Carl , Gerwert Klaus
Published: July 2002

The extremely slow α-helix/β-sheet transition of
proteins is a crucial step in amylogenic diseases and represents an
internal rearrangement of local contacts in an already folded protein.
These internal structural rearrangements within an already folded
protein are a critical aspect of biological action and are a product of
conformational flow along unknown metastable local minima of the energy
landscape of the compact protein. We use a diffusional IR mixer with
time-resolved Fourier transform IR spectroscopy capable of 400-μs
time resolution to show that the trifluoroethanol driven
β-sheet to α-helix transition of β-lactoglobulin proceeds via a
compact β-sheet intermediate with a lifetime of 7 ms, small compared
with the overall folding time of β-lactoglobulin.
Changing the lactose permease of
Escherichia coli
into a galactose-specific symporter
Authors: Guan Lan , Sahin-Tóth Miklós , Kaback H. Ronald
Published: July 2002

N
-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and β,
d
-galactopyranosyl 1-thio-β,
d
-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [
14
C]NEM. Remarkably, however,
d
-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consistently, competitive inhibition of [
14
C]galactose transport by lactose, melibiose, or β,
d
-galactopyranosyl 1-thio-β,
d
-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected. The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected. In addition, although the conservative mutation Ala-122 → Ser causes only mild inhibition of lactose transport, the mutations Ala-122 → Phe and Ala-122 → Tyr lead to marked inhibition. In contradistinction, none of these replacements has a marked effect on galactose transport. The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the nongalactosyl moiety of
d
-galactopyranosides. This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of
d
-galactopyranosides.
The c-Myc target gene
PRDX3
is required for mitochondrial homeostasis and neoplastic transformation
Authors: Wonsey Diane R. , Zeller Karen I. , Dang Chi V.
Published: July 2002

Deregulated expression of the c-Myc transcription factor is found in a wide variety of human tumors. Because of this significant role in oncogenesis, considerable effort has been devoted to elucidating the molecular program initiated by deregulated c-
myc
expression. The primary transforming activity of Myc is thought to arise through transcriptional regulation of numerous target genes. Thus far, Myc target genes involved in mitochondrial function have not been characterized in depth. Here, we describe a nuclear c-Myc target gene,
PRDX3
, which encodes a mitochondrial protein of the peroxiredoxin gene family. Expression of
PRDX3
is induced by the mycER system and is reduced in c-
myc
−/−
cells. Chromatin immunoprecipitation analysis spanning the entire
PRDX3
genomic sequence reveals that Myc binds preferentially to a 930-bp region surrounding exon 1. We show that
PRDX3
is required for Myc-mediated proliferation, transformation, and apoptosis after glucose withdrawal. Results using mitochondria-specific fluorescent probes demonstrate that
PRDX3
is essential for maintaining mitochondrial mass and membrane potential in transformed rat and human cells. These data provide evidence that
PRDX3
is a c-Myc target gene that is required to maintain normal mitochondrial function.
Energetics of glycerol conduction through aquaglyceroporin GlpF
Authors: Jensen Morten Ø. , Park Sanghyun , Tajkhorshid Emad , Schulten Klaus
Published: July 2002

Aquaglyceroporin GlpF selectively conducts water and linear polyalcohols, such as glycerol, across the inner membrane of
Escherichia coli
. We report steered molecular dynamics simulations of glycerol conduction through GlpF, in which external forces accelerate the transchannel conduction in a manner that preserves the intrinsic conduction mechanism. The simulations reveal channel-glycerol hydrogen bonding interactions and the stereoselectivity of the channel. Employing Jarzynski's identity between free energy and irreversible work, we reconstruct the potential of mean force along the conduction pathway through a time series analysis of molecular dynamics trajectories. This potential locates binding sites and barriers inside the channel; it also reveals a low energy periplasmic vestibule suited for efficient uptake of glycerol from the environment.
Squamous epithelial proliferation induced by walleye dermal sarcoma retrovirus cyclin in transgenic mice
Authors: Lairmore Michael D. , Stanley James R. , Weber Stacy A. , Holzschu Donald L.
Published: July 2002

Walleye dermal sarcoma (WDS) is a common disease of walleye fish in the United States and Canada. These proliferative lesions are present autumn through winter and regress in the spring. Walleye dermal sarcoma virus (WDSV), a retrovirus distantly related to other members of the family
Retroviridae
, has been etiologically linked to the development of WDS. We have reported that the D-cyclin homologue [retroviral (rv) cyclin] encoded by WDSV rescues yeast conditionally deficient for cyclin synthesis from growth arrest and that WDSV-cyclin mRNA is present in developing tumors. These data strongly suggest that the rv-cyclin plays a central role in the development of WDS. To test the ability of the WDSV rv-cyclin to induce cell proliferation, we have generated transgenic mice expressing the rv-cyclin in squamous epithelia from the bovine keratin-5 promoter. The transgenic animals were smaller than littermates, had reduced numbers of hair follicles, and transgenic females did not lactate properly. Following injury the transgenic animals developed severe squamous epithelial hyperplasia and dysplasia with ultrastructural characteristics of neoplastic squamous epithelium. Immunocytochemistry studies demonstrated that the hyperplastic epithelium stained positive for cytokeratin and were abnormally differentiated. Furthermore, the rv-cyclin protein was detected in the thickened basal cell layers of the proliferating lesions. These data are the first to indicate that the highly divergent WDSV rv-cyclin is a very potent stimulator of eukaryotic cell proliferation and to demonstrate the potential of a cyclin homologue encoded by a retrovirus to induce hyperplastic skin lesions.
CD2 molecules redistribute to the uropod during T cell scanning: Implications for cellular activation and immune surveillance
Authors: Tibaldi Elena V. , Salgia Ravi , Reinherz Ellis L.
Published: July 2002

Dynamic binding between CD2 and CD58 counter-receptors on opposing cells optimizes immune recognition through stabilization of cell–cell contact and juxtaposition of surface membranes at a distance suitable for T cell receptor–ligand interaction. Digitized time-lapse differential interference contrast and immunofluorescence microscopy on living cells now show that this binding also induces T cell polarization. Moreover, CD2 can facilitate motility of T cells along antigen-presenting cells via a movement referred to as scanning. Both activated CD4 and CD8 T cells are able to scan antigen-presenting cells surfaces in the absence of cognate antigen. Scanning is critically dependent on T cell β-integrin function, as well as myosin light chain kinase. More importantly, surface CD2 molecules rapidly redistribute on interaction with a cellular substratum, resulting in a 100-fold greater CD2 density in the uropod versus the leading edge. In contrast, no redistribution is observed for CD11a/CD18 or CD45. Molecular compartmentalization of CD2, T cell receptor, and lipid rafts within the uropod prearranges the cellular activation machinery for subsequent immune recognition. This “presynapse” formation on primed T cells will likely facilitate the antigen-dependent recognition capability required for efficient immune surveillance.
A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway
Authors: Marchetti Maria A. , Kumar Sanjay , Hartsuiker Edgar , Maftahi Mohamed , Carr Antony M. , Freyer Greg A. , Burhans William C. , Huberman Joel A.
Published: July 2002

The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood. Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast,
Schizosaccharomyces pombe
, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways. The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase ɛ) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition). These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.
Copper-catalyzed oxidation of the recombinant SHa(29–231) prion protein
Authors: Requena Jesús R. , Groth Darlene , Legname Giuseppe , Stadtman Earl R. , Prusiner Stanley B. , Levine Rodney L.
Published: July 2002

Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29–231). Using Cu
2+
/ascorbate, we oxidized SHaPrP(29–231)
in vitro
. Oxidation was demonstrated by liquid chromatography/mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29–101 region, which includes the histidine-containing octarepeats, is particularly affected by oxidation. Oxidation was time- and copper concentration-dependent and was evident with copper concentrations as low as 1 μM. Concomitant with oxidation, SHaPrP(29–231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37°C with a 6-fold molar excess of Cu
2+
. These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.
DNA delivery by phage as a strategy for encapsulating toroidal condensates of arbitrary size into liposomes
Authors: Lambert Olivier , Letellier Lucienne , Gelbart William M. , Rigaud Jean-Louis
Published: July 2002

We report a strategy for encapsulating and condensing DNA. When T5 phage binds to its membrane protein receptor, FhuA, its double stranded DNA (120,000 bp) is progressively released base pair after base pair in the surrounding medium. Using cryoelectron microscopy, we have visualized the structures formed after T5 phage DNA is released into neutral unilamellar proteoliposomes reconstituted with the receptor FhuA. In the presence of spermine, toroidal condensates of circumferentially wrapped DNA were formed. Most significantly, the sizes of these toroids were shown to vary, from 90 to 200 nm in their outer diameters, depending on the number of DNA stands transferred. We have also analyzed T5 DNA release in bulk solution containing the detergent-solubilized FhuA receptor. After DNA release in a spermine containing solution, huge DNA condensates with a diameter of about 300 nm were formed containing the DNAs from as many as 10–20 capsids. At alkaline pH, the condensates appeared as large hollow cylinders with a diameter of 200 nm and a height of 100–200 nm. Overall, the striking feature of our experiments is that, because of the progressive release of DNA from the phage capsid, the mechanism of toroid formation is fundamentally different from that in the classical studies in which highly dilute, “naked” DNA is condensed by direct addition of polyvalent cations; as a consequence, our method leads to toroids of arbitrary size.
Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins
Authors: Ansari Anjum , Kuznetsov Serguei V. , Shen Yiqing
Published: July 2002

Elucidating the mechanism of folding of polynucleotides depends on accurate estimates of free energy surfaces and a quantitative description of the kinetics of structure formation. Here, the kinetics of hairpin formation in single-stranded DNA are measured after a laser temperature jump. The kinetics are modeled as configurational diffusion on a free energy surface obtained from a statistical mechanical description of equilibrium melting profiles. The effective diffusion coefficient is found to be strongly temperature-dependent in the nucleation step as a result of formation of misfolded loops that do not lead to subsequent zipping. This simple system exhibits many of the features predicted from theoretical studies of protein folding, including a funnel-like energy surface with many folding pathways, trapping in misfolded conformations, and non-Arrhenius folding rates.
Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation
Authors: Poltoratsky Vladimir , Woo Caroline J. , Tippin Brigette , Martin Alberto , Goodman Myron F. , Scharff Matthew D.
Published: July 2002

High affinity antibodies are generated in mice and humans by means of somatic hypermutation (SHM) of variable (V) regions of Ig genes. Mutations with rates of 10−5–10−3per base pair per generation, about 106-fold above normal, are targeted primarily at V-region hot spots by unknown mechanisms. We have measured mRNA expression of DNA polymerases ι, η, and ζ by using cultured Burkitt's lymphoma (BL)2 cells. These cells exhibit 5–10-fold increases in heavy-chain V-region mutations targeted only predominantly to RGYW (R = A or G, Y = C or T, W = T or A) hot spots if costimulated with T cells and IgM crosslinking, the presumedin vivorequirements for SHM. An ∼4-fold increase pol ι mRNA occurs within 12 h when cocultured with T cells and surface IgM crosslinking. Induction of pols η and ζ occur with T cells, IgM crosslinking, or both stimuli. The fidelity of pol ι was measured at RGYW hot- and non-hot-spot sequences situated at nicks, gaps, and double-strand breaks. Pol ι formed T⋅G mispairs at a frequency of 10−2, consistent with SHM-generated C to T transitions, with a 3-fold increased error rate in hot- vs. non-hot-spot sequences for the single-nucleotide overhang. The T cell and IgM crosslinking-dependent induction of pol ι at 12 h may indicate an SHM “triggering” event has occurred. However, pols ι, η, and ζ are present under all conditions, suggesting that their presence is not sufficient to generate mutations because both T cell and IgM stimuli are required for SHM induction.