bluuebunny/crossref_metadata_2025 huggingface dataset:
Central electron temperature enhancements due to sawtooth stabilization during counter electron cyclotron current drive in Tokamak à Configuration Variable
Authors: Pietrzyk Z. A. , Angioni C. , Behn R. , Coda S. , Goodman T. P. , Henderson M. A. , Hofmann F. , Hogge J-P. , Moret J-M. , Pochelon A. , Reimerdes H. , Sauter O. , Weisen H.
Published: July 2002

In the Tokamak à Configuration Variable (TCV), the central electron temperature obtained in discharges with counter (CNTR) electron cyclotron current drive (ECCD) is larger than with CO-ECCD or electron cyclotron resonance heating (ECRH) alone. Comparison of experimental results with calculations by the transport code PRETOR [IAEA Technical Conference on Advances in Simulation and Models of Thermonuclear Plasmas. Montreal 142 (International Atomic Energy Agency, Vienna 1992)] indicates that sawtooth stabilization is responsible for the increased confinement time and the attendant twofold enhancement of the central temperature. Sawtooth stabilization is caused in turn by the central safety factor q0 rising above 1 for CNTR-ECCD; by contrast, the simulation results show that q0<1 in the sawtoothing CO-ECCD and ECRH cases.
Mechanism of oxygen plasma etching of polydimethyl siloxane films
Authors: Chou N. J. , Tang C. H. , Paraszczak J. , Babich E.
Published: July 2002

We have used x-ray photoelectron spectroscopy and multiple internal reflection infrared spectroscopy to study the etching of polysiloxane films in an rf oxygen plasma. The results indicate that a structurally strained SiO2 layer, ∼1 nm thick, is formed on the film exposed to oxygen plasma. For a given rf power input this oxidized overlayer recedes with the etching front in a steady state fashion, acting as a protective layer for the underlying polysiloxane film. The etching mechanism is effectively a combination of two competitive processes, namely, ion sputtering and oxidation.
Efficient generation of surface acoustic waves by thermoelasticity
Authors: Arnold W. , Betz B. , Hoffmann B.
Published: July 2002

Short laser pulses were used for generating surface acoustic waves (SAW’s) on optically opaque solids via the thermoelastic effect. The spatial and temporal shapes of the laser pulses were either a spot or a line of variable total width δ=2a with a Gaussian power distribution. Maximal efficiency is obtained when a=vRτ(vR: Rayleigh velocity, 2τ: total laser-pulse length). This condition is caused by interference effects between the individual frequency components radiating from the area of the thermoelastic source. If the intensity of the laser beam were 100% modulated at a given frequency, maximal efficiency would occur if kR=(2)1/2/a (kR: wave vector of the SAW’s).
Spatial location of electron trapping defects on silicon by scanning tunneling microscopy
Authors: Welland M. E. , Koch R. H.
Published: July 2002

Individual electron trapping sites in thermally oxidized silicon substrates have been observed using a scanning tunneling microscope (STM). The excellent spatial resolution of the STM allows single defects to be identified and located on the surface. The tunneling current near single trapping sites was observed to switch between two well defined values as the site occupation changed. The magnitude of the change in the current and spatial extent of the current fluctuations, typically 3 nm, is consistent with the calculated change in tunneling current from a single electron being trapped in the oxide.
Arabidopsis
gp91
phox
homologues
AtrbohD
and
AtrbohF
are required for accumulation of reactive oxygen intermediates in the plant defense response
Authors: Torres Miguel Angel , Dangl Jeffery L. , Jones Jonathan D. G.
Published: July 2002

Reactive oxygen intermediates (ROI) are strongly associated
with plant defense responses. The origin of these ROI has been
controversial.
Arabidopsis
respiratory burst oxidase
homologues (
rboh
genes) have been proposed to play a
role in ROI generation. We analyzed lines carrying
dSpm
insertions in the highly expressed
AtrbohD
and
AtrbohF
genes. Both are required for full ROI production
observed during incompatible interactions with the bacterial pathogen
Pseudomonas syringae
pv. tomato
DC3000(
avrRpm1
) and the oomycete parasite
Peronospora parasitica
. We also observed reduced
cell death, visualized by trypan blue stain and reduced electrolyte
leakage, in the
Atrboh
mutants after
DC3000(
avrRpm1
) inoculation. However, enhanced cell
death is observed after infection of mutant lines with
P.
parasitica
. Paradoxically, although
atrbohD
mutation eliminated the majority of total ROI production,
atrbohF
mutation exhibited the strongest effect on cell
death.
Amino acid runs in eukaryotic proteomes and disease associations
Authors: Karlin Samuel , Brocchieri Luciano , Bergman Aviv , Mrázek Jan , Gentles Andrew J.
Published: July 2002

We present a comparative proteome analysis of the five complete
eukaryotic genomes (human,
Drosophila melanogaster,
Caenorhabditis elegans, Saccharomyces cerevisiae,
Arabidopsis thaliana
), focusing on individual and multiple
amino acid runs, charge and hydrophobic runs. We found that
human proteins with multiple long runs are often associated with
diseases; these include long glutamine runs that induce neurological
disorders, various cancers, categories of leukemias (mostly involving
chromosomal translocations), and an abundance of Ca
2 +
and
K
+
channel proteins. Many human proteins with multiple runs
function in development and/or transcription regulation and are
Drosophila
homeotic homologs. A large number of these
proteins are expressed in the nervous system. More than 80% of
Drosophila
proteins with multiple runs seem to function
in transcription regulation. The most frequent amino acid runs in
Drosophila
sequences occur for glutamine, alanine, and
serine, whereas human sequences highlight glutamate, proline, and
leucine. The most frequent runs in yeast are of serine, glutamine, and
acidic residues. Compared with the other eukaryotic proteomes, amino
acid runs are significantly more abundant in the fly. This finding
might be interpreted in terms of innate differences in DNA-replication
processes, repair mechanisms, DNA-modification systems, and mutational
biases. There are striking differences in amino acid runs for
glutamine, asparagine, and leucine among the five proteomes.
Differential role of SLP-76 domains in T cell development and function
Authors: Kumar Lalit , Pivniouk Vadim , de la Fuente Miguel A. , Laouini Dhafer , Geha Raif S.
Published: July 2002

The adapter SLP-76 is essential for thymocyte development. SLP-76−/−mice were reconstituted with SLP-76 deletion mutant transgenes to examine the role of SLP-76 domains in T cell development and function. The N-terminal domain deletion mutant completely failed to restore thymocyte development. Mice reconstituted with Gads-binding site and SH2 domain deletion mutants had decreased thymic cellularity, impaired transition from double to single positive thymocytes, and decreased numbers of mature T cells in the spleen. Calcium mobilization and extracellular signal-regulated protein kinase activation were decreased in the Gads-binding site mutant but almost normal in the SH2 domain mutant. T cells from both mutants failed to proliferate following T cell antigen receptor ligation. Nevertheless, both mutants mounted partial cutaneous hypersensitivity responses and normal T cell dependent IgG1 antibody responses. These results indicate differential roles for SLP-76 domains in T cell development, proliferation and effector functions.
Tumor lysate-pulsed dendritic cells can elicit an effective antitumor immune response during early lymphoid recovery
Authors: Asavaroengchai W. , Kotera Y. , Mulé J. J.
Published: July 2002

Dendritic cells (DC) can serve to immunize the newborn immune system to foreign antigen. In a lymphopenic environment, naive T cells undergoing homeostasis-driven proliferation can acquire increased sensitivity to antigen stimulation. Here, we evaluated the capacity of DC to effectively prime the host immune system to elicit antitumor effects in the setting of early lymphoid reconstitution after bone marrow transplantation (BMT). Indeed, bone marrow-derived, cytokine-driven DC pulsed with whole tumor lysates (TP-DC) could, early on, prime a specific and long-lasting antitumor immune response, which mediated the rejection of a lethal challenge of a weakly immunogenic breast tumor. In the therapeutic setting, TP-DC could also inhibit the growth of preexisting breast tumor metastases by repetitive immunizations initiated early after BMT. Spleen T cells obtained from mice immunized with TP-DC early after BMT showed a substantial increase in tumor-specific IFN-γ production. Our findings demonstrate that it is possible to promote effective antitumor immunity in a defined lymphopenic environment through DC-based immunization.
Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies
Authors: Daugherty Patrick S. , Chen Gang , Iverson Brent L. , Georgiou George
Published: July 2002

Random mutagenesis and selection using phage or cell surface display provides an efficient method for affinity maturation of single chain Fv (scFv) antibodies, thereby improving function in various applications. To investigate the effects of mutation frequency on affinity maturation, error-prone PCR was used to generate libraries containing an average (
m
) of between 1.7 and 22.5 base substitutions per gene in a high affinity scFv antibody that binds to the cardiac glycoside digoxigenin. The scFv antibody libraries were displayed on
Escherichia coli
, and mutant populations were analyzed by flow cytometry. At low to moderate mutation frequencies with an average mutation rate of
m
≤ 8, the fraction of clones exhibiting binding to a fluorescently labeled conjugate of digoxigenin decreased exponentially (
r
2
= 0.99), but the most highly mutated library (
m
= 22.5) had significantly more active clones than expected relative to this trend. A library with a low error rate (
m
= 1.7), one with moderate error rate (
m
= 3.8), and the one with high error rate (
m
= 22.5) were screened for high affinity clones under conditions of identical stringency using fluorescence-activated cell sorting. After several rounds of enrichment, each of the three libraries yielded clones with improved affinity for the hapten. The moderate and high error rate libraries gave rise to clones exhibiting the greatest affinity improvement. Taken together, our results indicate that (
i
) functional clones occur at an unexpectedly high frequency in hypermutated libraries, (
ii
) gain-of-function mutants are well represented in such libraries, and (
iii
) the majority of the scFv mutations leading to higher affinity correspond to residues distant from the binding site.
A dynamic model of the type-2 inositol trisphosphate receptor
Authors: Sneyd James , Dufour Jean-François
Published: July 2002

The dynamic properties of the inositol (1,4,5)-trisphosphate (IP
3
) receptor are crucial for the control of intracellular Ca
2+
, including the generation of Ca
2+
oscillations and waves. However, many models of this receptor do not agree with recent experimental data on the dynamic responses of the receptor. We construct a model of the IP
3
receptor and fit the model to dynamic and steady-state experimental data from type-2 IP
3
receptors. Our results indicate that, (
i
) Ca
2+
binds to the receptor using saturating, not mass-action, kinetics; (
ii
) Ca
2+
decreases the rate of IP
3
binding while simultaneously
increasing
the steady-state sensitivity of the receptor to IP
3
; (
iii
) the rate of Ca
2+
-induced receptor activation increases with Ca
2+
and is faster than Ca
2+
-induced receptor inactivation; and (
iv
) IP
3
receptors are sequentially activated and inactivated by Ca
2+
even when IP
3
is bound. Our results emphasize that measurement of steady-state properties alone is insufficient to characterize the functional properties of the receptor.
Dynamic sensory sensitivity and children's word decoding skills
Authors: Talcott Joel B. , Witton Caroline , McLean Maggie F. , Hansen Peter C. , Rees Adrian , Green Gary G. R. , Stein John F.
Published: July 2002

The relationship between sensory sensitivity and reading performance was examined to test the hypothesis that the orthographic and phonological skills engaged in visual word recognition are constrained by the ability to detect dynamic visual and auditory events. A test battery using sensory psychophysics, psychometric tests, and measures of component literacy skills was administered to 32 unselected 10-year-old primary school children. The results suggest that children's sensitivity to both dynamic auditory and visual stimuli are related to their literacy skills. Importantly, after controlling for intelligence and overall reading ability, visual motion sensitivity explained independent variance in orthographic skill but not phonological ability, and auditory FM sensitivity covaried with phonological skill but not orthographic skill. These results support the hypothesis that sensitivity at detecting dynamic stimuli influences normal children's reading skills. Vision and audition separately may affect the ability to extract orthographic and phonological information during reading.
Molecules within molecules: Recognition through self-assembly
Authors: Hof Fraser , Rebek Julius
Published: July 2002

Synthetic molecular receptors that completely surround their target molecules can be created through the use of noncovalent interactions. These molecular capsules selectively sequester guest molecules from the influence of bulk solvent and other molecules on the basis of size, shape, and chemical complementarity. This reversible isolation spawns unique behavior within the confines of the host; the catalysis of chemical reactions and the stabilization of reactive species are possible outcomes that have been recently demonstrated. Compartmentalization of reagents can also have a dramatic effect on reactions that take place outside of the capsule, producing nonlinear kinetics in relatively simple reaction systems.
The brain-derived neurotrophic factor enhances synthesis of Arc in synaptoneurosomes
Authors: Yin Yong , Edelman Gerald M. , Vanderklish Peter W.
Published: July 2002

Protein synthesis in neurons is essential for the consolidation of memory and for the stabilization of activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP). Activity-dependent translation of dendritically localized mRNAs has been proposed to be a critical source of new proteins necessary for synaptic change. mRNA for the activity-regulated cytoskeletal protein, Arc, is transcribed during LTP and learning, and disruption of its translation gives rise to deficits in both. We have found that selective translation of Arc in a synaptoneurosomal preparation is induced by the brain-derived neurotrophic factor, a neurotrophin that is released during high-frequency stimulation patterns used to elicit LTP. This effect involves signaling through the TrkB receptor and is blocked by the
N
-methyl-
d
-aspartate-type glutamate receptor antagonist, MK801. The results suggest there is a synergy between neurotrophic and ionotropic mechanisms that may influence the specificity and duration of changes in synaptic efficacy at glutamatergic synapses.
Impaired retention of spatial memory after transection of longitudinally oriented axons of hippocampal CA3 pyramidal cells
Authors: Steffenach Hill-Aina , Sloviter Robert S. , Moser Edvard I. , Moser May-Britt
Published: July 2002

Longitudinally oriented axon collaterals of CA3 pyramidal cells may be critical for integrating distributed information in the hippocampus. To investigate the possible role of this pathway in the retention of spatial memory, we made a single transversely oriented cut through the dorsal CA3 region of each hippocampus. Although the lesion involved <3% of the hippocampal volume, it nonetheless disrupted memory retention in a water maze in preoperatively trained rats. New learning in a different water maze was attenuated. No significant impairment occurred in rats with longitudinally oriented cuts, or in animals with ibotenic acid-induced lesions of similar magnitude. To characterize the effect of a focal lesion on the integrity of longitudinally projecting axons, we stained degenerating cells and fibers in rats with unilateral CA3 transections by using FluoroJade-B. Degenerating terminals were seen across a wide region posterior to the cut, and were present in the strata of areas CA3 and CA1 that are innervated by CA3 pyramidal cells. These results suggest that the integrity of longitudinally oriented, translamellar axons of CA3 pyramidal cells may be necessary for efficient acquisition and retention of spatial memory.
Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein
Authors: Wang Lin , Sunahara Roger K. , Krumins Andrejs , Perkins Guy , Crochiere Marsha L. , Mackey Mason , Bell Sean , Ellisman Mark H. , Taylor Susan S.
Published: July 2002

Differential compartmentalization of signaling molecules in
cells and tissues is being recognized as an important mechanism for
regulating the specificity of signal transduction pathways. A kinase
anchoring proteins (AKAPs) direct the subcellular localization of
protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual
specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue
fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding
region and a putative regulator of G protein signaling (RGS) domain was
previously identified by means of a yeast two-hybrid screen. Here, we
report the cloning of full-length human D-AKAP2 (662 residues) with an
additional putative RGS domain, and the corresponding mouse protein
less the first two exons (617 residues). Expression of D-AKAP2 was
characterized by using mouse tissue extracts. Full-length D-AKAP2 from
various tissues shows different molecular weights, possibly because of
alternative splicing or posttranslational modifications. The cloned
human gene product has a molecular weight similar to one of the
prominent mouse proteins.
In vivo
association of D-AKAP2
with PKA in mouse brain was demonstrated by using cAMP agarose
pull-down assay. Subcellular localization for endogenous
mouse, rat, and human D-AKAP2 was determined by immunocytochemistry,
immunohistochemistry, and tissue fractionation. D-AKAP2 from all three
species is highly enriched in mitochondria. The mitochondrial
localization and the presence of RGS domains in D-AKAP2 may have
important implications for its function in PKA and G protein signal
transduction.
Plasticity of the cochleotopic (frequency) map in specialized and nonspecialized auditory cortices
Authors: Sakai Masashi , Suga Nobuo
Published: July 2002

Auditory conditioning (associative learning) causes reorganization
of the cochleotopic (frequency) maps of the primary auditory cortex
(AI) and the inferior colliculus. Focal electric stimulation of the AI
also evokes basically the same cortical and collicular reorganization
as that caused by conditioning. Therefore, part of the neural mechanism
for the plasticity of the central auditory system caused by
conditioning can be explored by focal electric stimulation of the AI.
The reorganization is due to shifts in best frequencies (BFs) together
with shifts in frequency-tuning curves of single neurons. In the AI of
the Mongolian gerbil (
Meriones unguiculatus
) and the
posterior division of the AI of the mustached bat (
Pteronotus
parnellii
), focal electric stimulation evokes BF shifts of
cortical auditory neurons located within a 0.7-mm distance along the
frequency axis. The amount and direction of BF shift differ depending
on the relationship in BF between stimulated and recorded neurons, and
between the gerbil and mustached bat. Comparison in BF shift between
different mammalian species and between different cortical areas of a
single species indicates that BF shift toward the BF of electrically
stimulated cortical neurons (centripetal BF shift) is common in the AI,
whereas BF shift away from the BF of electrically stimulated cortical
neurons (centrifugal BF shift) is special. Therefore, we propose a
hypothesis that reorganization, and accordingly organization, of
cortical auditory areas caused by associative learning can be quite
different between specialized and nonspecialized (ordinary) areas of
the auditory cortex.
Anion recognition as a method for templating pseudorotaxane formation
Authors: Wisner James A. , Beer Paul D. , Berry Neil G. , Tomapatanaget Boosayarat
Published: July 2002

Two thread-shaped cations, pyridinium nicotinamide and imidazolium, as their chloride and hexafluorophosphate salts, were studied with regards to complexation with hydrogen-bond-donating acyclic and macrocyclic ligands. In the latter case, the cations form pseudorotaxanes templated by the chloride anion but not hexafluorophosphate. This formation is a function of the coupling of ion-pairing between the cation and chloride anion and subsequent recognition of the anion by the macrocyclic diamide, which provides the driving force for interpenetration. We propose that this anion template principle is a general method for the construction of pseudorotaxanes and could be applied to other cationic threads, anions, and macrocyclic species.
Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from
Chrysanthemum cinerariaefolium
Authors: Rivera Susan B. , Swedlund Bradley D. , King Gretchen J. , Bell Russell N. , Hussey Charles E. , Shattuck-Eidens Donna M. , Wrobel Wislawa M. , Peiser Galen D. , Poulter C. Dale
Published: July 2002

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the
condensation of two molecules of dimethylallyl diphosphate to produce
chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail
or irregular c1′-2-3 linkage between isoprenoid units. Irregular
monoterpenes are common in
Chrysanthemum
cinerariaefolium
and related members of the Asteraceae family.
In
C. cinerariaefolium,
CPP is an intermediate in the
biosynthesis of the pyrethrin ester insecticides. CPPase was purified
from immature chrysanthemum flowers, and the N terminus of the protein
was sequenced. A
C. cinerariaefolium
λ cDNA library
was screened by using degenerate oligonucleotide probes based on the
amino acid sequence to identify a CPPase clone that encoded a 45-kDa
preprotein. The first 50 aa of the ORF constitute a putative plastidial
targeting sequence. Recombinant CPPase bearing an N-terminal
polyhistidine affinity tag in place of the targeting sequence was
purified to homogeneity from an overproducing
Escherichia
coli
strain by Ni
2+
chromatography. Incubation of
recombinant CPPase with dimethylallyl diphosphate produced CPP. The
diphosphate ester was hydrolyzed by alkaline phosphatase, and the
resulting monoterpene alcohol was analyzed by GC/MS to confirm
its structure. The amino acid sequence of CPPase aligns closely with
that of the chain elongation prenyltransferase farnesyl diphosphate
synthase rather than squalene synthase or phytoene synthase, which
catalyze c1′-2-3 cyclopropanation reactions similar to the CPPase
reaction.
Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection
Authors: Zhang Jianzhi , Dyer Kimberly D. , Rosenberg Helene F.
Published: July 2002

The mammalian RNase A superfamily comprises a diverse array of ribonucleolytic proteins that have a variety of biochemical activities and physiological functions. Two rapidly evolving RNases of higher primates are of particular interest as they are major secretory proteins of eosinophilic leukocytes and have been found to possess anti-pathogen activitiesin vitro. To understand how these RNases acquired this function during evolution and to develop animal models for the study of their functionsin vivo, it is necessary to investigate these genes in many species. Here, we report the sequences of 38 functional genes and 23 pseudogenes of the eosinophil-associated RNase (EAR) family from 5 rodent species. Our phylogenetic analysis of these genes showed a clear pattern of evolution by a rapid birth-and-death process and gene sorting, a process characterized by rapid gene duplication and deactivation occurring differentially among lineages. This process ultimately generates distinct or only partially overlapping inventories of the genes, even in closely related species. Positive Darwinian selection also contributed to the diversification of these EAR genes. The striking similarity between the evolutionary patterns of the EAR genes and those of the major histocompatibility complex, immunoglobulin, and T cell receptor genes stands in strong support of the hypothesis that host-defense and generation of diversity are among the primary physiological function of the rodent EARs. The discovery of a large number of divergent EARs suggests the intriguing possibility that these proteins have been specifically tailored to fight against distinct rodent pathogens.
Comparative genomic analysis in the region of a major
Plasmodium
-refractoriness locus of
Anopheles
gambiae
Authors: Thomasová Dana , Ton Lucas Q. , Copley Richard R. , Zdobnov Evgeny M. , Wang Xuelan , Hong Young S. , Sim Cheolho , Bork Peer , Kafatos Fotis C. , Collins Frank H.
Published: July 2002

We have sequenced six overlapping clones from a library of bacterial artificial chromosome (BAC) clones derived from a laboratory strain of the mosquito,
Anopheles gambiae
, the major vector of human malaria in Africa. The resulting uninterrupted 528-kb sequence is from the
8C
region of the mosquito
2R
chromosome, at or very near the major refractoriness locus associated with melanotic encapsulation of parasites. This sequence represents the first extensive view of the mosquito genome structure encompassing 48 genes. Genomic comparison reveals that the majority of the orthologues are found in six microsyntenic clusters in
Drosophila melanogaster
. A BAC clone that is wholly contained within this region demonstrates the existence of a remarkable degree of local polymorphism in this species, which may prove important for its population structure and vectorial capacity.
Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation
Authors: Endrizzi James A. , Beernink Peter T. , Alber Tom , Schachman H. K.
Published: July 2002

A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. For
Escherichia coli
aspartate transcarbamoylase (ATCase; EC
2.1.3.2
) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analog
N
-phosphonacetyl-
l
-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-Å resolution crystal structure of the C trimer–PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer–PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.
Differential regulation of the human and murine CD34 genes in hematopoietic stem cells
Authors: Okuno Yutaka , Iwasaki Hiromi , Huettner Claudia S. , Radomska Hanna S. , Gonzalez David A. , Tenen Daniel G. , Akashi Koichi
Published: July 2002

Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34
−/lo
express the hCD34 transgene. These data strongly support the notion that hCD34
+
human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.
Neurotrophin secretion from hippocampal neurons evoked by long-term-potentiation-inducing electrical stimulation patterns
Authors: Gärtner Annette , Staiger Volker
Published: July 2002

The neurotrophin (NT) brain-derived neurotrophic factor (BDNF) plays an essential role in the formation of long-term potentiation (LTP). Here, we address whether this modulation by BDNF requires its continuous presence, or whether a local increase in BDNF is necessary during a specific time period of LTP initiation. Using electrical field stimulation of primary cultures of hippocampal neurons, we demonstrate that short high-frequency bursts of stimuli that induce LTP evoke also an instantaneous secretion of BDNF. In contrast, stimuli at low frequencies, inducing long-term depression, do not enhance BDNF secretion, suggesting that BDNF is specifically present, and thus required, at the time of LTP induction. The field-stimulation-mediated BDNF secretion depends on the formation of action potentials and is induced by IP
3
-mediated Ca
2+
release from intracellular stores. Experiments, aimed at determining the sites of NT secretion that use NT6, showed similar patterns of surface labeling by field stimulation to those shown previously by high potassium.
Removing symbioticWolbachiabacteria specifically inhibits oogenesis in a parasitic wasp
Authors: Dedeine Franck , Vavre Fabrice , Fleury Frédéric , Loppin Benjamin , Hochberg Michael E. , Boulétreau Michel
Published: July 2002

Wolbachiaare bacteria that live in the cells of various invertebrate species to which they cause a wide range of effects on physiology and reproduction. We investigated the effect ofWolbachiainfection in the parasitic wasp,Asobara tabidaNees (Hymenoptera, Braconidae). In the 13 populations tested, all individuals proved to be infected byWolbachia. The removal ofWolbachiaby antibiotic treatment had a totally unexpected effect—aposymbiotic female wasps were completely incapable of producing mature oocytes and therefore could not reproduce. In contrast, oogenesis was not affected in treatedAsobara citri, a closely related species that does not harborWolbachia. No difference between natural symbiotic and cured individuals was found for other adult traits including male fertility, locomotor activity, and size, indicating that the effect on oogenesis is highly specific. We argue that indirect effects of the treatments used in our study (antibiotic toxicity or production of toxic agents) are very unlikely to explain the sterility of females, and we present results showing a direct relationship between oocyte production andWolbachiadensity in females. We conclude thatWolbachiais necessary for oogenesis in theseA. tabidastrains, and this association would seem to be the first example of a transition from facultative to obligatory symbiosis in arthropod–Wolbachiaassociations.
Reversible cardiac fibrosis and heart failure induced by conditional expression of an antisense mRNA of the mineralocorticoid receptor in cardiomyocytes
Authors: Beggah Ahmed T. , Escoubet Brigitte , Puttini Stefania , Cailmail Stephane , Delage Vanessa , Ouvrard-Pascaud Antoine , Bocchi Brigitte , Peuchmaur Michel , Delcayre Claude , Farman Nicolette , Jaisser Frederic
Published: July 2002

Cardiac failure is a common feature in the evolution of cardiac disease. Among the determinants of cardiac failure, the renin–angiotensin–aldosterone system has a central role, and antagonism of the mineralocorticoid receptor (MR) has been proposed as a therapeutic strategy. In this study, we questioned the role of the MR, not of aldosterone, on heart function, using an inducible and cardiac-specific transgenic mouse model. We have generated a conditional knock-down model by expressing solely in the heart an antisense mRNA directed against the murine MR, a transcription factor with unknown targets in cardiomyocytes. Within 2–3 mo, mice developed severe heart failure and cardiac fibrosis in the absence of hypertension or chronic hyperaldosteronism. Moreover, cardiac failure and fibrosis were fully reversible when MR antisense mRNA expression was subsequently suppressed.
Binding strength and activation state of single fibrinogen-integrin pairs on living cells
Authors: Litvinov Rustem I. , Shuman Henry , Bennett Joel S. , Weisel John W.
Published: July 2002

Integrin activation states determine the ability of these receptors to mediate cell–matrix and cell–cell interactions. The prototypic example of this phenomenon is the platelet integrin, αIIbβ3. In unstimulated platelets, αIIbβ3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables αIIbβ3 to bind ligands such as fibrinogen and von Willebrand factor. To study the regulation of integrin activation states at the level of single molecules, we developed a model system based on laser tweezers, enabling us to determine the rupture forces required to separate single ligand-receptor pairs by using either purified proteins or intact living cells. Here, we show that rupture forces of individual fibrinogen molecules and either purified αIIbβ3 or αIIbβ3 on the surface of living platelets were 60 to 150 pN with a peak yield strength of 80–100 pN. Platelet stimulation using either ADP or the thrombin receptor-activating peptide enhanced the accessibility but not the adhesion strength of single αIIbβ3 molecules, indicating that there are only two states of αIIbβ3 activation. Thus, we found it possible to use laser tweezers to measure the regulation of forces between individual ligand-receptor pairs on living cells. This methodology can be applied to the study of other regulated cell membrane receptors using the ligand-receptor yield strength as a direct measure of receptor activation/inactivation state.
The
Caenorhabditis elegans hif-1
gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia
Authors: Jiang Huaqi , Guo Rong , Powell-Coffman Jo Anne
Published: July 2002

Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Here, we present evidence that a similar complex mediates response to hypoxia in
Caenorhabditis elegans
. This complex consists of HIF-1 and AHA-1, which are encoded by
C. elegans
homologs of the hypoxia-inducible factor (HIF) α and β subunits, respectively.
hif-1
mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of
hif-1
-defective animals die in these conditions. We show that the expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both
hif-1
and
aha-1
are expressed in most cell types, and the gene products can be coimmunoprecipitated. We conclude that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, we find that nuclear localization of AHA-1 is disrupted in an
hif-1
mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1.
Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme
Authors: Yan Wei , Wu Faye , Morser John , Wu Qingyu
Published: July 2002

Atrial natriuretic peptide (ANP) is a cardiac hormone essential for the regulation of blood pressure. In cardiac myocytes, ANP is synthesized as a precursor, pro-ANP, that is converted to biologically active ANP by an unknown membrane-associated protease. Recently, we cloned a transmembrane serine protease, corin, that is highly expressed in the heart. In this study, we examine effects of corin on pro-ANP processing. Our results show that recombinant human corin converts pro-ANP to ANP and that the cleavage in pro-ANP by corin is highly sequence specific. Our findings suggest that corin is the long-sought pro-ANP-converting enzyme and that the corin-mediated pro-ANP activation may play a role in regulating blood pressure.
Patterns of DNA sequence polymorphism along chromosome 1 of maize (
Zea mays
ssp.
mays
L.)
Authors: Tenaillon Maud I. , Sawkins Mark C. , Long Anthony D. , Gaut Rebecca L. , Doebley John F. , Gaut Brandon S.
Published: July 2002

We measured sequence diversity in 21 loci distributed along chromosome 1 of maize (
Zea mays
ssp.
mays
L.). For each locus, we sequenced a common sample of 25 individuals representing 16 exotic landraces and nine U.S. inbred lines. The data indicated that maize has an average of one single nucleotide polymorphism (SNP) every 104 bp between two randomly sampled sequences, a level of diversity higher than that of either humans or
Drosophila melanogaster
. A comparison of genetic diversity between the landrace and inbred samples showed that inbreds retained 77% of the level of diversity of landraces, on average. In addition, Tajima's
D
values suggest that the frequency distribution of polymorphisms in inbreds was skewed toward fewer rare variants. Tests for selection were applied to all loci, and deviations from neutrality were detected in three loci. Sequence diversity was heterogeneous among loci, but there was no pattern of diversity along the genetic map of chromosome 1. Nonetheless, diversity was correlated (
r
= 0.65) with sequence-based estimates of the recombination rate. Recombination in our sample was sufficient to break down linkage disequilibrium among SNPs. Intragenic linkage disequilibrium declines within 100–200 bp on average, suggesting that genome-wide surveys for association analyses require SNPs every 100–200 bp.
Scan statistics to scan markers for susceptibility genes
Authors: Hoh J. , Ott J.
Published: July 2002

Scan statistics are applied to combine information on multiple contiguous genetic markers used in a genome screen for susceptibility loci. This information may be, for example, allele sharing proportions for sib pairs or logarithm of odds (lod) scores in general small families. We focus on a dichotomous outcome variable, for example, case and control individuals or affected-affected versus affected-unaffected siblings, and suitable single-marker statistics. A significant scan statistic based on the single-marker statistics represents evidence of the presence of a susceptibility gene. For a given length of the scan statistic, we assess its significance by Monte Carlo permutation tests. Comparing
P
values for varying lengths of scan statistics, we treat the smallest observed
P
value as our statistic of interest and determine its overall significance level. We applied this method to a genome screen with autism families. The result was informative and surprising: A susceptibility region was found (genome-wide significance level,
P
= 0.038), which is missed with conventional approaches.
Crossmodal binding of fear in voice and face
Authors: Dolan R. J. , Morris J. S. , de Gelder B.
Published: July 2002

In social environments, multiple sensory channels are simultaneously engaged in the service of communication. In this experiment, we were concerned with defining the neuronal mechanisms for a perceptual bias in processing simultaneously presented emotional voices and faces. Specifically, we were interested in how bimodal presentation of a fearful voice facilitates recognition of fearful facial expression. By using event-related functional MRI, that crossed sensory modality (visual or auditory) with emotional expression (fearful or happy), we show that perceptual facilitation during face fear processing is expressed through modulation of neuronal responses in the amygdala and the fusiform cortex. These data suggest that the amygdala is important for emotional crossmodal sensory convergence with the associated perceptual bias during fear processing, being mediated by task-related modulation of face-processing regions of fusiform cortex.
Speech comprehension is correlated with temporal response patterns recorded from auditory cortex
Authors: Ahissar Ehud , Nagarajan Srikantan , Ahissar Merav , Protopapas Athanassios , Mahncke Henry , Merzenich Michael M.
Published: July 2002

Speech comprehension depends on the integrity of both the spectral content and temporal envelope of the speech signal. Although neural processing underlying spectral analysis has been intensively studied, less is known about the processing of temporal information. Most of speech information conveyed by the temporal envelope is confined to frequencies below 16 Hz, frequencies that roughly match spontaneous and evoked modulation rates of primary auditory cortex neurons. To test the importance of cortical modulation rates for speech processing, we manipulated the frequency of the temporal envelope of speech sentences and tested the effect on both speech comprehension and cortical activity. Magnetoencephalographic signals from the auditory cortices of human subjects were recorded while they were performing a speech comprehension task. The test sentences used in this task were compressed in time. Speech comprehension was degraded when sentence stimuli were presented in more rapid (more compressed) forms. We found that the average comprehension level, at each compression, correlated with (
i
) the similarity between the frequencies of the temporal envelopes of the stimulus and the subject's cortical activity (“stimulus-cortex frequency-matching”) and (
ii
) the phase-locking (PL) between the two temporal envelopes (“stimulus-cortex PL”). Of these two correlates, PL was significantly more indicative for single-trial success. Our results suggest that the match between the speech rate and the
a priori
modulation capacities of the auditory cortex is a prerequisite for comprehension. However, this is not sufficient: stimulus-cortex PL should be achieved during actual sentence presentation.
Neuronal representations of stimulus associations develop in the temporal lobe during learning
Authors: Messinger Adam , Squire Larry R. , Zola Stuart M. , Albright Thomas D.
Published: July 2002

Visual stimuli that are frequently seen together become associated in long-term memory, such that the sight of one stimulus readily brings to mind the thought or image of the other. It has been hypothesized that acquisition of such long-term associative memories proceeds via the strengthening of connections between neurons representing the associated stimuli, such that a neuron initially responding only to one stimulus of an associated pair eventually comes to respond to both. Consistent with this hypothesis, studies have demonstrated that individual neurons in the primate inferior temporal cortex tend to exhibit similar responses to pairs of visual stimuli that have become behaviorally associated. In the present study, we investigated the role of these areas in the formation of conditional visual associations by monitoring the responses of individual neurons during the learning of new stimulus pairs. We found that many neurons in both area TE and perirhinal cortex came to elicit more similar neuronal responses to paired stimuli as learning proceeded. Moreover, these neuronal response changes were learning-dependent and proceeded with an average time course that paralleled learning. This experience-dependent plasticity of sensory representations in the cerebral cortex may underlie the learning of associations between objects.
THEMATICS: A simple computational predictor of enzyme function from structure
Authors: Ondrechen Mary Jo , Clifton James G. , Ringe Dagmar
Published: July 2002

We show that theoretical microscopic titration curves (THEMATICS) can be used to identify active-site residues in proteins of known structure. Results are featured for three enzymes: triosephosphate isomerase (TIM), aldose reductase (AR), and phosphomannose isomerase (PMI). We note that TIM and AR have similar structures but catalyze different kinds of reactions, whereas TIM and PMI have different structures but catalyze similar reactions. Analysis of the theoretical microscopic titration curves for all of the ionizable residues of these proteins shows that a small fraction (3–7%) of the curves possess a flat region where the residue is partially protonated over a wide pH range. The preponderance of residues with such perturbed curves occur in the active site. Additional results are given in summary form to show the success of the method for proteins with a variety of different chemistries and structures.
Spontaneous hepatocellular carcinoma is reduced in transgenic mice overexpressing human
O
6
- methylguanine-DNA methyltransferase
Authors: Zhou Zi-Qiang , Manguino Diwi , Kewitt Kristen , Intano Gabriel W. , McMahan C. Alex , Herbert Damon C. , Hanes Martha , Reddick Robert , Ikeno Yuji , Walter Christi A.
Published: July 2002

O
6
-methylguanine (
O
6
mG) is a potent mutagenic and procarcinogenic DNA lesion. Organisms have evolved with a DNA repair mechanism that largely ameliorates the deleterious effects of
O
6
mG through a direct reversal mechanism by a protein termed
O
6
-methylguanine-DNA methyltransferase (MGMT). However, the contribution of
O
6
mG to carcinogenesis, in the absence of known exposure to agents that produce it, has not been defined. Nontransgenic C3HeB male mice have a high frequency of spontaneous liver tumors. Transgenic CeHeB/FeJ mice expressing human MGMT (hMGMT) were generated that had elevated hepatic MGMT activity. The spontaneous development of hepatocellular carcinoma was significantly reduced in those mice expressing hMGMT compared with nontransgenic C3HeB/FeJ male mice. No differences were detected in spontaneous mutant frequencies in
lacI
transgenes in mice carrying hMGMT compared with that without hMGMT but the proportion of GC to AT transition mutations was lower in the transgenic mice carrying hMGMT as well as
lacI
. Tumors that arose in C3HeB/FeJ transgenic mice were largely deficient in hMGMT protein as determined by immunohistochemistry with a monoclonal antibody directed against hMGMT. Together these data indicate that spontaneous
O
6
mG lesions induced hepatocellular carcinogenesis in C3HeB/FeJ male mice. These transgenic mice represent a rare example of reduced spontaneous carcinogenesis.