bluuebunny/crossref_metadata_2025 huggingface dataset:
Reaction of SiO2 with hafnium oxide in low oxygen pressure
Authors: Wang S. J. , Lim P. C. , Huan A. C. H. , Liu C. L. , Chai J. W. , Chow S. Y. , Pan J. S. , Li Q. , Ong C. K.
Published: March 2003

A dynamic process consisting of a series of reactions during deposition of HfO2 films on SiO2-covered silicon under oxygen-deficient conditions is identified. The oxygen-deficient HfOx<2 layer absorbs the oxygen in the SiO2 layer to form fully oxidized metal oxide film. As a result, there is no silicate and silicon oxide formed at the interface with silicon substrate. Thermodynamic analysis indicates that even if there is a layer of silicate forming at the initial stage of deposition, the silicate layer will be decomposed with the progress of HfOx<2 deposition.
Observation of metal–insulator transition in Al–Pd–Re quasicrystals by x-ray absorption and photoemission spectroscopy
Authors: Lay Y. Y. , Jan J. C. , Chiou J. W. , Tsai H. M. , Pong W. F. , Tsai M.-H. , Pi T. W. , Lee J. F. , Ma C. I. , Tseng K. L. , Wang C. R. , Lin S. T.
Published: March 2003

Using x-ray absorption and valence-band photoelectron spectroscopy (PES) we investigated the electronic structures of icosahedral (i)-Al70Pd22.5Re7.5 quasicrystals (QCs) with a 4.2 K/300 K resistivity ratio, r, ranging from 8.3 to 107 obtained under various annealing conditions. Our Al K- and Pd L3-edge x-ray absorption results show that the density of states, N(E), near the Fermi level, EF, jumps to a larger value when r decreases down to below about 20.6. The valence-band PES results show that N(E) near EF is greatly reduced in i-Al70Pd22.5Re7.5 QCs relative to that of the pure metal, which confirms the existence of the pseudogap. The PES spectrum has a sharp cutoff at EF for r=8.3 QC, while it decreases smoothly down to zero at EF for larger r’s. The combined results suggest the occurrence of metal–insulator transition at an r between 13 and 20.6.
Intracrystalline monitoring of molecular uptake into the one-dimensional channels of the AFI-type crystals using interference microscopy
Authors: Lehmann Enrico , Vasenkov Sergey , Kärger Jörg , Zadrozna Gabriela , Kornatowski Jan
Published: March 2003

Intracrystalline concentration profiles of methanol during its adsorption into the one-dimensional channels of CrAPO-5 crystals are reported. The profiles were recorded by the interference microscopy technique recently introduced in our laboratory. The exceptionally high spatial resolution, which can be achieved by this technique allowed us to obtain detailed information on the interplay of intracrystalline diffusion, the permeability of the crystal surface and the role of the intergrowth effects on molecule uptake. Comparison of the measured concentration profiles with those simulated by the dynamic Monte Carlo method provides quantitative information on the intracrystalline diffusivity of methanol and the permeability of the transport barriers on the crystal surface.
Time-resolved cathodoluminescence assessment of deep-level transitions in hydride-vapor-phase-epitaxy GaN
Authors: Dı́az-Guerra C. , Piqueras J. , Cavallini A.
Published: March 2003

The temporal behavior of deep-level luminescence emissions in undoped hydride-vapor-phase-epitaxy GaN layers of different thicknesses has been investigated by time-resolved cathodoluminescence (TRCL). The complex nature of the yellow luminescence is revealed in the TRCL spectra by the presence of two bands peaked at 2.22 and 2.03 eV. A red band with a decay time of 700 μs, centered at about 1.85 eV, dominates spectra recorded for long delay times. Exponential transients with associated decay times of hundreds of μs were measured at 87 K for all the deep-level emissions found in the layers.
Dynamics of nanoscale polarization backswitching in tetragonal lead zirconate titanate thin film
Authors: Fu Desheng , Suzuki Kazuyuki , Kato Kazumi , Suzuki Hisao
Published: March 2003

The dynamics of polarization backswitching in highly oriented Pb(Zr0.3Ti0.7)O3 (PZT) thin films has been studied in nanoscale using piezoresponse scanning force microscopy (SFM). Our measurements reveal that a SFM-written domain with diameter about 200 nm in PZT films loses its polarization through both the sidewise and forward backswitching. Both of these sidewise and forward domain wall motions follow a stretched exponential law. However, the characteristic time τ of forward backswitching are about ten times of that sidewise motion. The time dependence of sidewise domain wall velocity indicates that there is a time dependence of activation energy of the barrier that domain motions encounter.
Effect of a rotating electrode on the formation of single-walled carbon nanotubes
Authors: Bae Jun Cheol , Yoon Young Joon , Baik Hong Koo , Lee Se-Jong , Song Kie Moon
Published: March 2003

We investigated the effect of a rotating anode on the formation of single-walled carbon nanotubes (SWNTs) using the plasma rotating electrode process. With rotation of the anode, the diameter distribution of SWNTs was shifted toward smaller diameters, and the yield of SWNTs increased. In addition, optical emission spectroscopy confirmed the increase of plasma temperature by rotating anode. These results indicate that the diameter distribution of SWNTs and the increase of SWNT yield resulted from the change of discharge characteristics by rotation of the anode.
Microwave properties of compositionally graded (Ba, Sr)TiO3 thin films according to the direction of the composition gradient for tunable microwave applications
Authors: Lee Su-Jae , Moon Seung Eon , Ryu Han-Cheol , Kwak Min-Hwan , Kim Young-Tae , Han Seok-Kil
Published: March 2003

Compositionally graded (Bax, Sr1−x)TiO3 (BST) thin films were deposited on MgO substrates by pulsed laser ablation. The microwave properties of the graded BST thin films were investigated at microwave frequencies with coplanar waveguide (CPW) meander-line phase shifters as a function of the direction of the composition gradient with respect to the substrate at room temperature. CPW phase shifters using graded BaTiO3(BT)→SrTiO3(ST) and ST→BT thin films exhibited a differential phase shift of 73° and 22° at 10 GHz with a dc bias voltage of 150 V, respectively. The figure of merit of the phase shifters at 10 GHz was 14.6 deg/dB for the graded BT→ST film, and 10 deg/dB for the graded ST→BT film. The graded BT→ST thin films showed a larger phase tuning than the ST→BT thin films. The microwave properties of the graded BST films depended strongly on the crystalline structure and the direction of the composition gradient with respect to the substrate.
Control of tunnel oxide thickness in Si-nanocrystal array memories obtained by ion implantation and its impact in writing speed and volatility
Authors: González-Varona O. , Garrido B. , Cheylan S. , Pérez-Rodrı́guez A. , Cuadras A. , Morante J. R.
Published: March 2003

The injection and storage of charge in Si nanocrystals obtained by ion implantation and annealing have been studied for different tunnel oxide thicknesses. The energy of the ions was kept fixed at 15 keV, which is compatible with most ion implanters used in Si technology, and the distance between the Si nanocrystals and the substrate was controlled by using gate oxides with different thicknesses. The processing conditions were adjusted for precipitating all the Si excess and for having Si–SiO2 interfaces free of defects. Consequently, reliable structures were obtained working in the direct tunneling injection regime, which show unprecedented longer retention times. Furthermore, it is shown that by changing only the oxide thickness it is possible to engineer devices with a tradeoff between writing speed and retention time.
Connecting statistical and optimized potentials in protein folding via a generalized foldability criterion
Authors: Saven Jeffery G.
Published: March 2003

Developing a predictive understanding of protein folding requires quantitative measures of sequence-structure compatibility. A folding criterion is presented whose optimization over a training set of structures yields the commonly used statistical and optimized potentials as two limiting cases, whereas these potentials are more often inferred from databases using two very different approaches. A statistical potential is shown to be a form of optimized potential within the context of an independent pair approximation.
Insulin-degrading enzyme regulates the levels of insulin, amyloid β-protein, and the β-amyloid precursor protein intracellular domain
in vivo
Authors: Farris Wesley , Mansourian Stefan , Chang Yang , Lindsley Loren , Eckman Elizabeth A. , Frosch Matthew P. , Eckman Christopher B. , Tanzi Rudolph E. , Selkoe Dennis J. , Guénette Suzanne
Published: April 2003

Two substrates of insulin-degrading enzyme (IDE), amyloid β-protein (Aβ) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Aβ levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Aβ and insulin degradation
in vivo
and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE −/−). IDE deficiency resulted in a >50% decrease in Aβ degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE −/− mice showed increased cerebral accumulation of endogenous Aβ, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the β-amyloid precursor protein, which was recently found to be degraded by IDE
in vitro
. Together with emerging genetic evidence, our
in vivo
findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD.
Statistically relevant conserved quantities for truncated quasigeostrophic flow
Authors: Abramov Rafail V. , Majda Andrew J.
Published: April 2003

Systematic applications of ideas from equilibrium statistical mechanics lead to promising strategies for assessing the unresolved scales of motion in many problems in science and engineering. A scientific debate over more than the last 25 years involves which conserved quantities among the formally infinite list are statistically relevant for the large-scale equilibrium statistical behavior. Here this important issue is addressed by using suitable discrete numerical approximations for geophysical flows with many conserved quantities as a numerical laboratory. The results of numerical experiments are presented here for these truncated geophysical flows with topography in a suitable regime. These experiments establish that the integrated third power of potential vorticity besides the familiar constraints of energy, circulation, and enstrophy (the integrated second power) is statistically relevant in this regime for the coarse-grained equilibrium statistical behavior at large scales. Furthermore, the integrated higher powers of potential vorticity larger than three are statistically irrelevant for the large-scale equilibrium statistical behavior in the examples studied here.
Phosphatidylinositol 4-kinase type IIα is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles
Authors: Guo Jun , Wenk Markus R. , Pellegrini Lorenzo , Onofri Franco , Benfenati Fabio , De Camilli Pietro
Published: April 2003

Phosphorylation of inositol phospholipids plays a key role in cellular regulation via the generation of intracellular second messengers. In addition, it represents a mechanism to regulate interactions of the lipid bilayer with proteins and protein scaffolds involved in vesicle budding, cytoskeletal organization, and signaling. Generation of phosphatidylinositol 4-phosphate [PI(4)P] from phosphatidylinositol (PI) is an important step in this metabolic pathway because PI(4)P is a precursor of other important phosphoinositides and has protein binding properties of its own. We report here that a PI 4-kinase (PI4K) activity previously reported on synaptic vesicles is accounted for by the α isoform of the recently characterized type II PI4K (PI4KII) family. PI4KIIα, which also accounts for the bulk of PI4K activity in brain extracts, is concentrated at synapses and in the region of the Golgi complex in neuronal perikarya. Our results provide new evidence for the occurrence of a cycle of phosphoinositide synthesis and hydrolysis nested within the exo–endocytic cycle of synaptic vesicles and point to PI4KIIα as a critical player in this cycle.
Sequence information can be obtained from single DNA molecules
Authors: Braslavsky Ido , Hebert Benedict , Kartalov Emil , Quake Stephen R.
Published: April 2003

The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.
Neuron recognition by parallel Potts segmentation
Authors: Peng S. , Urbanc B. , Cruz L. , Hyman B. T. , Stanley H. E.
Published: April 2003

Identifying neurons and their spatial coordinates in images of the cerebral cortex is a necessary step in the quantitative analysis of spatial organization in the brain. This is especially important in the study of Alzheimer's disease (AD), in which spatial neuronal organization and relationships are highly disrupted because of neuronal loss. To automate neuron recognition by using high-resolution confocal microscope images from human brain tissue, we propose a recognition method based on statistical physics that consists of image preprocessing, parallel image segmentation, and cluster selection on the basis of shape, optical density, and size. We segment a preprocessed digital image into clusters by applying Monte Carlo simulations of a
q
-state inhomogeneous Potts model. We then select the range of Potts segmentation parameters to yield an ideal recognition of simplified objects in the test image. We apply our parallel segmentation method to control individuals and to AD patients and achieve recognition of 98% (for a control) and 93% (for an AD patient), with at most 3% false clusters.
Cloning and engineering of the cinnamycin biosynthetic gene cluster from
Streptomyces cinnamoneus cinnamoneus
DSM 40005
Authors: Widdick D. A. , Dodd H. M. , Barraille P. , White J. , Stein T. H. , Chater K. F. , Gasson M. J. , Bibb M. J.
Published: April 2003

Lantibiotics are ribosomally synthesized oligopeptide antibiotics that contain lanthionine bridges derived by the posttranslational modification of amino acid residues. Here, we describe the cinnamycin biosynthetic gene cluster (
cin
) from
Streptomyces cinnamoneus cinnamoneus
DSM 40005, the first, to our knowledge, lantibiotic gene cluster from a high G+C bacterium to be cloned and sequenced. The
cin
cluster contains many genes not found in lantibiotic clusters from low G+C Gram-positive bacteria, including a
Streptomyces
antibiotic regulatory protein regulatory gene, and lacks others found in such clusters, such as a LanT-type transporter and a LanP-type protease. Transfer of the
cin
cluster to
Streptomyces lividans
resulted in heterologous production of cinnamycin. Furthermore, modification of the cinnamycin structural gene (
cinA
) led to production of two naturally occurring lantibiotics, duramycin and duramycin B, closely resembling cinnamycin, whereas attempts to make a more widely diverged derivative, duramycin C, failed to generate biologically active material. These results provide a basis for future attempts to construct extensive libraries of cinnamycin variants.
Pitx3 is required for development of substantia nigra dopaminergic neurons
Authors: Nunes Irene , Tovmasian Lucy T. , Silva Robert M. , Burke Robert E. , Goff Stephen P.
Published: April 2003

Dopaminergic (DA) neurons of substantia nigra in the midbrain control voluntary movement, and their degeneration is the cause of Parkinson's disease. The complete set of genes required to specifically determine the development of midbrain DA subgroups is not known yet. We report here that mice lacking the
bicoid
-related homeoprotein Pitx3 fail to develop DA neurons of the substantia nigra. Other mesencephalic DA neurons of the ventral tegmental area and retrorubral field are unaltered in their dopamine expression and histological organization. These data suggest that Pitx3-dependent gene expression is specifically required for the differentiation of DA progenitors within the mesencephalic DA system.
Cell and tumor classification using gene expression data: Construction of forests
Authors: Zhang Heping , Yu Chang-Yung , Singer Burton
Published: April 2003

The advent of gene chips has led to a promising technology for cell, tumor, and cancer classification. We exploit and expand the methodology of recursive partitioning trees for tumor and cell classification from microarray gene expression data. To improve classification and prediction accuracy, we introduce a deterministic procedure to form forests of classification trees and compare their performance with extant alternatives. When two published and commonly used data sets are used, we find that the deterministic forests perform similarly to the random forests in terms of the error rate obtained from the leave-one-out procedure, and all of the forests are far better than the single trees. In addition, we provide graphical presentations to facilitate interpretation of complex forests and compare our findings with the current biological literature. In addition to numerical improvement, the main advantage of deterministic forests is reproducibility and scientific interpretability of all steps in tree construction.
Real-time semantic compensation in patients with agrammatic comprehension: Electrophysiological evidence for multiple-route plasticity
Authors: Hagoort Peter , Wassenaar Marlies , Brown Colin
Published: April 2003

To understand spoken language requires that the brain provides rapid access to different kinds of knowledge, including the sounds and meanings of words, and syntax. Syntax specifies constraints on combining words in a grammatically well formed manner. Agrammatic patients are deficient in their ability to use these constraints, due to a lesion in the perisylvian area of the language-dominant hemisphere. We report a study on real-time auditory sentence processing in agrammatic comprehenders, examining their ability to accommodate damage to the language system. We recorded event-related brain potentials (ERPs) in agrammatic comprehenders, nonagrammatic aphasics, and age-matched controls. When listening to sentences with grammatical violations, the agrammatic aphasics did not show the same syntax-related ERP effect as the two other subject groups. Instead, the waveforms of the agrammatic aphasics were dominated by a meaning-related ERP effect, presumably reflecting their attempts to achieve understanding by the use of semantic constraints. These data demonstrate that although agrammatic aphasics are impaired in their ability to exploit syntactic information in real time, they can reduce the consequences of a syntactic deficit by exploiting a semantic route. They thus provide evidence for the compensation of a syntactic deficit by a stronger reliance on another route in mapping sound onto meaning. This is a form of plasticity that we refer to as multiple-route plasticity.
Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3-spectratype analysis
Authors: Hofbauer Monika , Wiesener Solveigh , Babbe Holger , Roers Axel , Wekerle Hartmut , Dornmair Klaus , Hohlfeld Reinhard , Goebels Norbert
Published: April 2003

Clonal expansions of CD8+ T cells have been identified in muscle and blood of polymyositis patients by PCR techniques, including T cell receptor (TCR) complementarity-determining region (CDR)3 length analysis (spectratyping). To examine a possible pathogenic role of these clonally expanded T cells, we combined CDR3 spectratyping with laser microdissection and single-cell PCR of individual myocytotoxic T cells that contact, invade, and destroy a skeletal muscle fiber. First, we screened cDNA from muscle biopsy specimens by CDR3 spectratyping for expanded TCR β chain variable region (BV) sequences. To pinpoint the corresponding T cells in tissue, we stained cryostat sections with appropriate anti-TCR BV mAbs, isolated single BV+ T cells that directly contacted or invaded a muscle fiber by laser-assisted microdissection, and amplified their TCR BV chain sequences from rearranged genomic DNA. In this way, we could relate the oligoclonal peaks identified by CDR3-spectratype screening to morphologically characterized microdissected T cells. In one patient, a large fraction of the microdissected T cells carried a common TCR-BV amino acid CDR3 motif and conservative nucleotide exchanges in the CDR3 region, suggesting an antigen-driven response. In several cases, we tracked these T cell clones for several years in CD8+ (but not CD4+) blood lymphocytes and in two patients also in consecutive muscle biopsy specimens. During immunosuppressive therapy, oligoclonal CDR3-spectratype patterns tended to revert to more polyclonal Gaussian distribution-like patterns. Our findings demonstrate that CDR3 spectratyping and single-cell analysis can be combined to identify and track autoaggressive T cell clones in blood and target tissue. This approach should be applicable to other inflammatory and autoimmune disorders.
Dietary zinc modulates gene expression in murine thymus: Results from a comprehensive differential display screening
Authors: Moore J. Bernadette , Blanchard Raymond K. , Cousins Robert J.
Published: April 2003

Differential mRNA display was used to comprehensively screen the murine thymic transcriptome for genes modulated
in vivo
by dietary zinc. A moderate feeding protocol rendered young adult, outbred mice zinc-deficient and zinc-supplemented without alterations in feeding behavior or growth. However, these levels of deficiency and supplementation altered specific mRNA abundances in a manner detectable by differential display. In total, 240 primer-pair combinations were used to generate >48,000 interpretable cDNA bands derived from thymic total RNA, of which only 265 or 0.55% were identified as zinc-modulated under these moderate dietary conditions. The most strongly zinc-modulated cDNAs identified by display were reamplified and sequenced. No cDNAs encoding zinc-metalloenzymes or zinc-finger transcription factors were identified as zinc-modulated in this global screening. Those zinc-regulated genes independently confirmed by quantitative PCR included: heat shock proteins 40 and 60; heat shock cognate 70; histocompatibility 2, class II antigen A, α; and the T cell cytokine receptor. In addition, a variety of transcription- and translation-related factors (such as ribosomal proteins L3, L5, and L28; nuclear matrix protein 84; matrin cyclophilin; the H3 histone family 3A protein; β
2
microglobulin; and a cleavage and polyadenylation factor) were identified as zinc-modulated. These profiling data show that differential expression of genes in the thymus in response to the dietary zinc supply precedes many of the phenotypic effects on thymic function associated with severe zinc restriction or supplementation. Several genes involved in T cell development were identified as regulated by zinc and will be targets to evaluate the effects of zinc on immune function.
Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis
Authors: Chabes Anna Lena , Pfleger Cathie M. , Kirschner Marc W. , Thelander Lars
Published: April 2003

Ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, and catalyzes the rate-limiting step in DNA precursor synthesis: the reduction of ribonucleotides to deoxyribonucleotides. A strictly balanced supply of deoxyribonucleotides is essential for both accurate DNA replication and repair. Therefore, ribonucleotide reductase activity is under exquisite control both transcriptionally and posttranscriptionally. In proliferating mammalian cells, enzyme activity is regulated by control of R2 protein stability. This control, which responds to DNA damage, is effective until cells pass into mitosis. We demonstrate that the mitotic degradation and hence the overall periodicity of R2 protein levels depends on a KEN box sequence, recognized by the Cdh1–anaphase-promoting complex. The mouse R2 protein specifically binds Cdh1 and is polyubiquitinated in an
in vitro
ubiquitin assay system. Mutating the KEN signal stabilizes the R2 protein during mitosis/G
1
in R2 protein-overexpressing cells. The degradation process, which blocks deoxyribonucleotide production during G
1
, may be an important mechanism protecting the cell against unscheduled DNA synthesis. The newly discovered p53-induced p53R2 protein that lacks a KEN box may supply deoxyribonucleotides for DNA repair during G
0
/G
1
.
Hg(II) binding to a weakly associated coiled coil nucleates an encoded metalloprotein fold: A kinetic analysis
Authors: Farrer Brian T. , Pecoraro Vincent L.
Published: April 2003

A detailed kinetic analysis of metal encapsulation by a
de novo
-designed protein is described. The kinetic mechanism of Hg(II) encapsulation in the three-stranded coiled coil formed by the peptide CH
3
CO-G LKALEEK CKALEEK LKALEEK G-NH
2
(Baby L9C) is derived by global analysis. The mechanism involves rapid initial collapse of two peptides by Hg(II) forming Hg(Baby L9C
-H
)
2
with a linear thiolato Hg(II) bound to the cysteine sulfur atoms. Here, Baby L9C
-H
denotes Baby L9C with the cysteine thiol deprotonated. Addition of the third peptide, forming the three-stranded coiled coil, is the rate-determining step and results in an intermediate state involving two separate species. One of the species, termed the properly folded intermediate, undergoes rapid deprotonation of the third cysteine thiol, yielding the desired three-stranded coiled coil with an encapsulated trigonal thiolato Hg(II). The other species, termed the misfolded intermediate, rearranges in an experimentally distinguishable step to the properly folded intermediate. The order of the reaction involving the addition of the third peptide with respect to the concentration of Baby L9C indicates that addition of the third helix only occurs through reaction of Hg(Baby L9C
-H
)
2
and Baby L9C that is unassociated with a coiled coil. Temperature dependence of the reaction afforded activation parameters for both the addition of the third helix (Δ
H
‡
= 20(2) kcal/mol; Δ
S
‡
= 40(5) cal/mol K) and the rearrangement of the misfolded intermediate steps (Δ
H
‡
= 23(2) kcal/mol; Δ
S
‡
= 27(5) cal/mol K). The mechanism is discussed with regard to metalloprotein folding and metalloprotein design.
Non-heme iron enzymes: Contrasts to heme catalysis
Authors: Solomon Edward I. , Decker Andrea , Lehnert Nicolai
Published: April 2003

Non-heme iron enzymes catalyze a wide range of O
2
reactions, paralleling those of heme systems. Non-heme iron active sites are, however, much more difficult to study because they do not exhibit the intense spectral features characteristic of the porphyrin ligand. A spectroscopic methodology was developed that provides significant mechanistic insight into the reactivity of non-heme ferrous active sites. These studies reveal a general mechanistic strategy used by these enzymes and differences in substrate and cofactor interactions dependent on their requirement for activation by iron. Contributions to O
2
activation have been elucidated for non-heme relative to heme ligand sets, and major differences in reactivity are defined with respect to the heterolytic and homolytic cleavage of O—O bonds.
Mammalian basal metabolic rate is proportional to body mass2/3
Authors: White Craig R. , Seymour Roger S.
Published: April 2003

The relationship between mammalian basal metabolic rate (BMR, ml of O2per h) and body mass (M, g) has been the subject of regular investigation for over a century. Typically, the relationship is expressed as an allometric equation of the form BMR =aMb. The scaling exponent (b) is a point of contention throughout this body of literature, within which arguments for and against geometric (b= 2/3) and quarter-power (b= 3/4) scaling are made and rebutted. Recently, interest in the topic has been revived by published explanations for quarter-power scaling based on fractal nutrient supply networks and four-dimensional biology. Here, a new analysis of the allometry of mammalian BMR that accounts for variation associated with body temperature, digestive state, and phylogeny finds no support for a metabolic scaling exponent of 3/4. Data encompassing five orders of magnitude variation inMand featuring 619 species from 19 mammalian orders show that BMR ∝M2/3.
Interdomain zinc site on human albumin
Authors: Stewart Alan J. , Blindauer Claudia A. , Berezenko Stephen , Sleep Darrell , Sadler Peter J.
Published: April 2003

Albumin is the major transport protein in blood for Zn
2+
, a metal ion required for physiological processes and recruited by various drugs and toxins. However, the Zn
2+
-binding site(s) on albumin is ill-defined. We have analyzed the 18 x-ray crystal structures of human albumin in the PDB and identified a potential five-coordinate Zn site at the interface of domains I and II consisting of N ligands from His-67 and His-247 and O ligands from Asn-99, Asp-249, and H
2
O, which are the same amino acid ligands as those in the zinc enzymes calcineurin, endonucleotidase, and purple acid phosphatase. The site is preformed in unliganded apo-albumin and highly conserved in mammalian albumins. We have used
111
Cd NMR as a probe for Zn
2+
binding to recombinant human albumin. We show that His-67 → Ala (His67Ala) mutation strongly perturbs Cd
2+
binding, whereas the mutations Cys34Ala, or His39Leu and Tyr84Phe (residues which may H-bond to Cys-34) have no effect. Weak Cl
−
binding to the fifth coordination site of Cd
2+
was demonstrated. Cd
2+
binding was dramatically affected by high fatty acid loading of albumin. Analysis of the x-ray structures suggests that fatty acid binding to site 2 triggers a spring-lock mechanism, which disengages the upper (His-67/Asn-99) and lower (His-247/Asp-249) halves of the metal site. These findings provide a possible mechanism whereby fatty acids (and perhaps other small molecules) could influence the transport and delivery of zinc in blood.
Canonical and unconventional pairing schemes between bis(nucleobase) complexes oftrans-a2PtII: Artificial nucleobase quartets and C—H…N bonds
Authors: Freisinger Eva , Rother Irene B. , Lüth Marc Sven , Lippert Bernhard
Published: April 2003

If two nucleobases are crosslinked bytrans-a2PtII, self-association via H bonding may take place either through individual bases or jointly through both bases. Due to the blockage of an acceptor site by the metal, the number of feasible pairing patterns can be reduced, and the preferred ones altered. If the metalated base pair as a whole undergoes association, base quartets can form. Various scenarios resulting from the application of guanine, hypoxanthine, and cytosine model nucleobases are discussed. Unconventional C—H…N hydrogen bonding has been observed in several instances.
Functional copper at the acetyl-CoA synthase active site
Authors: Seravalli Javier , Gu Weiwei , Tam Annie , Strauss Erick , Begley Tadhg P. , Cramer Stephen P. , Ragsdale Stephen W.
Published: April 2003

The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood–Ljungdahl pathway of autotrophic CO
2
fixation. A recent structure of the
Moorella thermoacetica
enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the
M. thermoacetica
ACS active site is essential. Depletion of copper correlates with reduction in ACS activity and in intensity of the “NiFeC” EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH. In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity. Cu is also found in the methanogenic CODH/ACS from
Methanosarcina thermophila
. XAS studies are consistent with a distorted Cu(I)–S
3
site in the fully active enzyme in solution. Cu extended x-ray absorption fine structure analysis indicates an average Cu–S bond length of 2.25 Å and a metal neighbor at 2.65 Å, consistent with the Cu–Ni distance observed in the crystal structure. XAS experiments in the presence of seleno-CoA reveal a Cu–S
3
Se environment with a 2.4-Å Se–Cu bond, strongly implicating a Cu–SCoA intermediate in the mechanism of acetyl-CoA synthesis. These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms.
Critical role of oxidative stress in estrogen-induced carcinogenesis
Authors: Bhat Hari K. , Calaf Gloria , Hei Tom K. , Loya Theresa , Vadgama Jaydutt V.
Published: April 2003

Mechanisms of estrogen-induced tumorigenesis in the target organ are not well understood. It has been suggested that oxidative stress resulting from metabolic activation of carcinogenic estrogens plays a critical role in estrogen-induced carcinogenesis. We tested this hypothesis by using an estrogen-induced hamster renal tumor model, a well established animal model of hormonal carcinogenesis. Hamsters were implanted with 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethinylestradiol (αEE), menadione, a combination of αE2 and αEE, or a combination of αEE and menadione for 7 months. The group treated with βE2 developed target organ specific kidney tumors. The kidneys of hamsters treated with αE2, αEE, or menadione alone did not show any gross evidence of tumor. Kidneys of hamsters treated with a combination of αE2 and αEE showed early signs of proliferation in the interstitial cells. Kidneys of hamsters treated with a combination of menadione and αEE showed foci of tumor with congested tubules and atrophic glomeruli. βE2-treated tumor-bearing kidneys showed >2-fold increase in 8-iso-prostaglandin F
2α
(8-iso-PGF
2α
) levels compared with untreated controls. Kidneys of hamsters treated with a combination of menadione and αEE showed increased 8-iso-PGF
2α
levels compared with untreated controls, whereas no increase in 8-iso-PGF
2α
was detected in kidneys of αEE-treated group. A chemical known to produce oxidative stress or a potent estrogen with poor ability to produce oxidative stress, were nontumorigenic in hamsters, when given as single agents, but induced renal tumors, when given together. Thus, these data provide evidence that oxidant stress plays a crucial role in estrogen-induced carcinogenesis.
Recombination signal sequence-binding protein Jκ alters mesodermal cell fate decisions by suppressing cardiomyogenesis
Authors: Schroeder Timm , Fraser Stuart T. , Ogawa Minetaro , Nishikawa Satomi , Oka Chio , Bornkamm Georg W. , Nishikawa Shin-Ichi , Honjo Tasuku , Just Ursula
Published: April 2003

The transcription factor recombination signal sequence-binding protein Jκ (RBP-J) is a key downstream element in the signaling pathway of all four mammalian Notch receptors that are critically involved in the control of embryonic and adult development. RBP-J-deficient mice display complex defects and die around day 9.5 postcoitum. Here, we investigate the function of RBP-J in the development of mesodermal cell lineages by using the OP9 stroma coculture system. RBP-J-deficient embryonic stem (ES) cells gave rise to cardiomyocytes, endothelial cells, and primitive and definitive hematopoietic cells. Thus, RBP-J-mediated signals are not required for generation of these cell types. However, when compared with parental RBP-J-expressing ES cells, cardiomyogenesis derived from RBP-J-deficient ES cells was increased. Repression over the cardiogenic pathway was restored by expressing RBP-J in RBP-J-deficient ES cells. Our data indicate that Notch signaling via RBP-J plays an important role for the correct specification of myocardial cell fates.
Control of steroid, heme, and carcinogen metabolism by nuclear pregnane X receptor and constitutive androstane receptor
Authors: Xie Wen , Yeuh Mei-Fei , Radominska-Pandya Anna , Saini Simrat P. S. , Negishi Yoichi , Bottroff Bobbie Sue , Cabrera Geraldine Y. , Tukey Robert H. , Evans Ronald M.
Published: April 2003

Through a multiplex promoter spanning 218 kb, the phase II UDP-glucuronosyltransferase 1A (
UGT1
) gene encodes at least eight differently regulated mRNAs whose protein products function as the principal means to eliminate a vast array of steroids, heme metabolites, environmental toxins, and drugs. The orphan nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) were originally identified as sensors able to respond to numerous environmentally derived foreign compounds (xenobiotics) to promote detoxification by phase I cytochrome P450 genes. In this report, we show that both receptors can induce specific UGT1A isoforms including those involved in estrogen, thyroxin, bilirubin, and carcinogen metabolism. Transgenic mice expressing a constitutively active form of human PXR show markedly increased UGT activity toward steroid, heme, and carcinogens, enhanced bilirubin clearance, as well as massively increased steroid clearance. The ability of PXR and constitutive androstane receptor and their ligands to transduce both the phase I and phase II adaptive hepatic response defines a unique transcriptional interface that bridges the ingestion and metabolism of environmental compounds to body physiology.
Regulation of Dlx3 gene expression in visceral arches by evolutionarily conserved enhancer elements
Authors: Sumiyama Kenta , Ruddle Frank H.
Published: April 2003

The mammalian Distal-less (Dlx) clusters (Dlx1-2, Dlx5-6, and Dlx3-7) have a nested expression pattern in developing visceral (branchial) arches. Genetic regulatory mechanisms controlling Dlx spatial expression within the visceral arches have not yet been defined. Here we show that an enhancer in the Dlx3-7 cluster can regulate the visceral arch specific expression pattern of the Dlx3 gene. We have used a 79-kb transgene construct containing the entire Dlx3-7 bigene cluster with a LacZ reporter inserted in frame in the first exon of the Dlx3 gene. Visceral arch expression is absent when a 4-kb element located within the Dlx3-7 intergenic region is deleted. A 245-bp element (I37-2) whose DNA sequence is highly conserved between human and mouse located within the 4kb-deleted region can drive visceral arch expression when fused to a hsp68-lacZ reporter transgene construct. Reporter expression is detected in 9.5 and 10.5 days postcoitum transgenic embryos in a manner consistent with the endogenous Dlx3 expression pattern in the mesenchyme of the first and second visceral arches. Thus the I37-2 element is both necessary and sufficient for Dlx3 expression. The I37-2 element contains several putative binding sites for several transcription factors including Dlx and other homeodomain proteins within the evolutionarily conserved region. Significantly, the I37-2 element shows a sequence-match including a Dlx binding site to a cis-element in the Dlx5-6 intermediate region designated mI56i [Zerucha, T., Stuhmer, T., Hatch, G., Park, B. K., Long, Q., Yu, G., Gambarotta, A., Schultz, J. R., Rubenstein, J. L. & Ekker, M. (2000)
J. Neurosci.
20, 709–721], despite distant phylogenetic relationship between these clusters. Our results provide evidence for a concerted role for DLX auto- and cross-regulation in the establishment of a nested expression pattern for Dlx3-7 and Dlx5-6 clusters within the visceral arches.
Essential function of α-calcium/calmodulin-dependent protein kinase II in neurotransmitter release at a glutamatergic central synapse
Authors: Hinds Heather L. , Goussakov Ivan , Nakazawa Kazu , Tonegawa Susumu , Bolshakov Vadim Y.
Published: April 2003

A significant fraction of the total calcium/calmodulin-dependent protein kinase II (CaMKII) activity in neurons is associated with synaptic connections and is present in nerve terminals, thus suggesting a role for CaMKII in neurotransmitter release. To determine whether CaMKII regulates neurotransmitter release, we generated and analyzed knockout mice in which the dominant α-isoform of CaMKII was specifically deleted from the presynaptic side of the CA3-CA1 hippocampal synapse. Conditional CA3 α-CaMKII knockout mice exhibited an unchanged basal probability of neurotransmitter release at CA3-CA1 synapses but showed a significant enhancement in the activity-dependent increase in probability of release during repetitive presynaptic stimulation, as was shown with the analysis of unitary synaptic currents. These data indicate that α-CaMKII serves as a negative activity-dependent regulator of neurotransmitter release at hippocampal synapses and maintains synapses in an optimal range of release probabilities necessary for normal synaptic operation.
ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery
Authors: Gao Hongbo , Kadirjan-Kalbach Deena , Froehlich John E. , Osteryoung Katherine W.
Published: April 2003

Chloroplast division in plant cells is orchestrated by a complex macromolecular machine with components positioned on both the inner and outer envelope surfaces. The only plastid division proteins identified to date are of endosymbiotic origin and are localized inside the organelle. Employing positional cloning methods in

Arabidopsis

in conjunction with a novel strategy for pinpointing the mutant locus, we have identified a gene encoding a new chloroplast division protein, ARC5. Mutants of
ARC5
exhibit defects in chloroplast constriction, have enlarged, dumbbell-shaped chloroplasts, and are rescued by a wild-type copy of
ARC5
. The
ARC5
gene product shares similarity with the dynamin family of GTPases, which mediate endocytosis, mitochondrial division, and other organellar fission and fusion events in eukaryotes. Phylogenetic analysis showed that ARC5 is related to a group of dynamin-like proteins unique to plants. A GFP–ARC5 fusion protein localizes to a ring at the chloroplast division site. Chloroplast import and protease protection assays indicate that the ARC5 ring is positioned on the outer surface of the chloroplast. Thus, ARC5 is the first cytosolic component of the chloroplast division complex to be identified. ARC5 has no obvious counterparts in prokaryotes, suggesting that it evolved from a dynamin-related protein present in the eukaryotic ancestor of plants. These results indicate that the chloroplast division apparatus is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes.
Prospective identification of tumorigenic breast cancer cells
Authors: Al-Hajj Muhammad , Wicha Max S. , Benito-Hernandez Adalberto , Morrison Sean J. , Clarke Michael F.
Published: April 2003

Breast cancer is the most common malignancy in United States women, accounting for >40,000 deaths each year. These breast tumors are comprised of phenotypically diverse populations of breast cancer cells. Using a model in which human breast cancer cells were grown in immunocompromised mice, we found that only a minority of breast cancer cells had the ability to form new tumors. We were able to distinguish the tumorigenic (tumor initiating) from the nontumorigenic cancer cells based on cell surface marker expression. We prospectively identified and isolated the tumorigenic cells as CD44
+
CD24
−/low
Lineage
−
in eight of nine patients. As few as 100 cells with this phenotype were able to form tumors in mice, whereas tens of thousands of cells with alternate phenotypes failed to form tumors. The tumorigenic subpopulation could be serially passaged: each time cells within this population generated new tumors containing additional CD44
+
CD24
−/low
Lineage
−
tumorigenic cells as well as the phenotypically diverse mixed populations of nontumorigenic cells present in the initial tumor. The ability to prospectively identify tumorigenic cancer cells will facilitate the elucidation of pathways that regulate their growth and survival. Furthermore, because these cells drive tumor development, strategies designed to target this population may lead to more effective therapies.
Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth
Authors: Leong Paul L. , Andrews Genevieve A. , Johnson Daniel E. , Dyer Kevin F. , Xi Sichuan , Mai Jeffrey C. , Robbins Paul D. , Gadiparthi Seshu , Burke Nancy A. , Watkins Simon F. , Grandis Jennifer Rubin
Published: April 2003

The transcription factor
s
ignal
t
ransducer and
a
ctivator of
t
ranscription 3 (Stat3) is constitutively activated in a variety of cancers including squamous cell carcinoma of the head and neck (SCCHN). Previous investigations have demonstrated that activated Stat3 contributes to a loss of growth control and transformation. To investigate the therapeutic potential of blocking Stat3 in cancer cells, we developed a transcription factor decoy to selectively abrogate activated Stat3. The Stat3 decoy was composed of a 15-mer double-stranded oligonucleotide, which corresponded closely to the Stat3 response element within the c-fos promoter. The Stat3 decoy bound specifically to activated Stat3 and blocked binding of Stat3 to a radiolabeled Stat3 binding element. By contrast, a mutated version of the decoy that differed by only a single base pair did not bind the activated Stat3 protein. Treatment of head and neck cancer cells with the Stat3 decoy inhibited proliferation and Stat3-mediated gene expression, but did not decrease the proliferation of normal oral keratinocytes. Thus, disruption of activated Stat3 by using a transcription factor decoy approach may serve as a novel therapeutic strategy for cancers characterized by constitutive Stat3 activation.