bluuebunny/crossref_metadata_2025 huggingface dataset:
Two-dimensional metastable magnetic semiconductor structures
Authors: Kolodziejski L. A. , Gunshor R. L. , Otsuka N. , Gu B. P. , Hefetz Y. , Nurmikko A. V.
Published: July 2002

Metastable zinc-blende MnSe has been grown by molecular beam epitaxy. The magnetic semiconductor has been incorporated into three novel superlattice structures which include a binary ZnSe/MnSe superlattice, a comb superlattice, and a ZnSe/(Zn,Mn)Se superlattice structure consisting of ZnSe wells perturbed by the insertion of ultrathin layers (two to three monolayers) of MnSe. Reflection high-energy electron diffraction and transmission electron microscopy reveal the zinc-blende crystal structure of the MnSe layers. Preliminary optical measurements show agreement with anticipated exciton emission energies; such spectra show large magnetic field induced shifts indicating that, although MnSe is antiferromagnetic, these ‘‘two-dimensional’’ magnetic sheets exhibit paramagnetic behavior.
Raman scattering from AlGaInP
Authors: Kondow Masahiko , Minagawa Shigekazu , Satoh Shin
Published: July 2002

Raman scattering spectra from AlGaInP quaternary alloys grown on GaAs substrates are measured. Dominant peaks observed are identified as AlP-, GaP-, and InP-like longitudinal optical modes. Mode frequency is seen to change almost linearly with aluminum composition, exhibiting the ‘‘partly three-mode type’’ behavior.
Low-threshold operation of AlGaAs/GaAs multiple quantum well lasers grown on Si substrates by molecular beam epitaxy
Authors: Chong Tow C. , Fonstad Clifton G.
Published: July 2002

We report the first AlGaAs-GaAs multiple quantum well (MQW) lasers fabricated on (001)Si substrates by molecular beam epitaxy (MBE) with relatively thin buffer layers. The lasers have broad-area (170×240 μm) pulsed threshold current density of 3.2 kA/cm2 at room temperature, the lowest ever reported for GaAs lasers grown by MBE on Si. Stripe lasers exhibit external differential quantum efficiencies comparable to those of GaAs lasers grown on GaAs substrates. Clean single longitudinal mode oscillations are observed, with transverse electric field the dominant polarization. The lasers also exhibit well-behaved fast pulse response, with a measured carrier lifetime of ∼2.2 ns in the active region. The improved performance is attributed to the employment of graded-index MQW structures and improved growth techniques.
Image processing by four-wave mixing in photorefractive GaAs
Authors: Gheen Gregory , Cheng Li-Jen
Published: July 2002

Three image processing experiments were performed by degenerate four-wave mixing in photorefractive GaAs. The experiments were imaging by phase conjugation, edge enhancement, and autocorrelation. The results show that undoped, semi-insulating, liquid-encapsulated Czochralski-grown GaAs crystals can be used as effective optical processing media despite their small electro-optic coefficient.
Electron accumulation layer at n-Si/non-liquid electrolyte interfaces
Authors: Benisty H. , Colomban Ph. , Chazalviel J.-N.
Published: July 2002

n-Si/non-liquid electrolyte interfaces have been realized in order to obtain electron accumulation layers of very high concentration at various temperatures. The interface has been characterized by using standard electrochemical measurements and the two-dimensional electron gas has been studied by transconductance and infrared optical absorption measurements. These interfaces are found to exhibit densities as high as 4×1013 electrons cm−2. These results are compared with available data on analogous n-Si/organic-solvent electrolyte interfaces.
Broadly tunable yellow-green laser using color centers in a LiF crystal at room temperature
Authors: Gu Hongen , Qi Lan , Wan Liangfeng
Published: July 2002

A broadly tunable yellow-green laser using F+3 centers and some unknown centers in a LiF crystal at room temperature is reported. The laser output extended from 500 to 640 nm and peaked at 535 and 610 nm.
Chemical etching of Y-Cu-Ba-O thin films
Authors: Shih I. , Qiu C. X.
Published: July 2002

Thin films of Y-Ba-Cu-O have been deposited by rf sputtering using a stoichiometric target on alumina, zirconia, and Y2O3 coated silicon substrates. After a brief heat treatment in oxygen, a resistive transition was observed with an onset temperature from 89 to 94.6 K. Etching experiments of the as-deposited films have been made to determine the etching rates of the compound. It was found that reproducible results can be readily obtained using the following solutions: H3PO4/H2O, HNO3/H2O, and HCl/H2O. Using a positive photoresist technology, 3 μm lines have been successfully produced by etching in these solutions. The present patterning process was found to have no deleterious effect on the superconducting characteristics of the Y-Ba-Cu-O films.
Experience-dependent plasticity without long-term depression by type 2 metabotropic glutamate receptors in developing visual cortex
Authors: Renger John J. , Hartman Kenichi N. , Tsuchimoto Yoshiko , Yokoi Mineto , Nakanishi Shigetada , Hensch Takao K.
Published: July 2002

Synaptic depression is thought to underlie the loss of cortical responsiveness to an eye deprived of vision. Here, we establish a fundamental role for type 2 metabotropic glutamate receptors (mGluR2) in long-term depression (LTD) of synaptic transmission within primary visual cortex. Direct mGluR2 activation by (2
S
,2′
R
,3′
R
-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) persistently depressed layer 2/3 field potentials in slices of mouse binocular zone when stimulated concomitantly. Chemical LTD was independent of
N
-methyl-
d
-aspartate (NMDA) receptors but occluded conventional LTD by low-frequency stimulation, indicating shared downstream events. Antagonists or targeted disruption of mGluR2 conversely prevented LTD induction by electrical low-frequency stimulation to layer 4. In contrast, Schaeffer collateral synapses did not exhibit chemical LTD, revealing hippocampal area CA1, naturally devoid of mGluR2, to be an inappropriate model for neocortical plasticity. Moreover, monocular deprivation remained effective in mice lacking mGluR2, and receptor expression levels were unchanged during the critical period in wild-type mice, indicating that experience-dependent plasticity is independent of LTD induction in visual cortex. Short-term depression that was unaffected by mGluR2 deletion may better reflect circuit refinement
in vivo
.
Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity
Authors: Khoshnan Ali , Ko Jan , Patterson Paul H.
Published: July 2002

We have generated eight mAbs (MW1–8) that bind the epitopes polyglutamine (polyQ), polyproline (polyP), or the C terminus of exon 1 in huntingtin (htt) protein. In the brains of Huntington's disease (HD) mouse models, the anti-polyQ mAbs bind to various cytoplasmic compartments, whereas the anti-polyP and anti-C terminus mAbs bind nuclear inclusions containing htt. To use these mAbs as intracellular perturbation agents, we have cloned and expressed the antigen-binding domains of three of the mAbs as single-chain variable region fragment Abs (scFvs). In 293 cells cotransfected with htt exon 1 containing an expanded polyQ domain, MW1, MW2, and MW7 scFvs colocalize with htt exon 1. Moreover, these scFvs coimmunoprecipitate with htt exon 1 in cell extracts. In perturbation experiments, MW7 scFv, recognizing the polyP domains of htt, significantly inhibits aggregation as well as the cell death induced by mutant htt protein. In contrast, MW1 and MW2 scFvs, recognizing the polyQ stretch, stimulate htt aggregation and apoptosis. Therefore, these anti-htt scFvs can be used to investigate the role of the polyP and polyQ domains in HD pathogenesis, and antibody binding to the polyP domain has potential therapeutic value in HD.
Large extinctions in an evolutionary model: The role of innovation and keystone species
Authors: Jain Sanjay , Krishna Sandeep
Published: July 2002

The causes of major and rapid transitions observed in biological macroevolution as well as in the evolution of social systems are a subject of much debate. Here we identify the proximate causes of crashes and recoveries that arise dynamically in a model system in which populations of (molecular) species coevolve with their network of chemical interactions. Crashes are events that involve the rapid extinction of many species, and recoveries the assimilation of new ones. These are analyzed and classified in terms of the structural properties of the network. We find that in the absence of large external perturbation, “innovation” is a major cause of large extinctions and the prime cause of recoveries. Another major cause of crashes is the extinction of a “keystone species.” Different classes of causes produce crashes of different characteristic sizes.
Analysis of Cdc6 function in the assembly of mammalian prereplication complexes
Authors: Cook Jeanette Gowen , Park Chi-Hyun , Burke Thomas W. , Leone Gustavo , DeGregori James , Engel Amber , Nevins Joseph R.
Published: July 2002

Eukaryotic DNA replication requires the previous formation of a prereplication complex containing the ATPase Cdc6 and the minichromosome maintenance (Mcm) complex. Although considerable insight has been gained from
in vitro
studies and yeast genetics, the functional analysis of replication proteins in intact mammalian cells has been lacking. We have made use of adenoviral vectors to express normal and mutant forms of Cdc6 in quiescent mammalian cells to assess function. We demonstrate that Cdc6 expression alone is sufficient to induce a stable association of endogenous Mcm proteins with chromatin in serum-deprived cells where cyclin-dependent kinase (cdk) activity is low. Moreover, endogenous Cdc6 is sufficient to load Mcm proteins onto chromatin in the absence of cdk activity in p21-arrested cells. Cdc6 synergizes with physiological levels of cyclin E/Cdk2 to induce semiconservative DNA replication in quiescent cells whereas cyclin A/Cdk2 is unable to collaborate with Cdc6. Cdc6 that cannot be phosphorylated by cdks is fully capable of inducing Mcm chromatin association and replication. Mutation of the Cdc6 ATP-binding site severely impairs the ability of Cdc6 to induce Mcm chromatin loading and reduces its ability to induce replication. Nevertheless, the ATPase domain of Cdc6 in the absence of the noncatalytic amino terminus is not sufficient for either Mcm chromatin loading or DNA replication, indicating a requirement for this domain of Cdc6.
Stop codons preceded by rare arginine codons are efficient determinants of SsrA tagging in
Escherichia coli
Authors: Hayes Christopher S. , Bose Baundauna , Sauer Robert T.
Published: July 2002

The SsrA or tmRNA quality control system intervenes when ribosomes stall on mRNAs and directs the addition of a C-terminal peptide tag that targets the modified polypeptide for degradation. Although hundreds of SsrA-tagged proteins can be detected in cells when degradation is prevented, most of these species have not been identified. Consequently, the mRNA sequence determinants that cause ribosome stalling and SsrA tagging are poorly understood. SsrA tagging of
Escherichia coli
ribokinase occurs at three specific sites at or near the C terminus of this protein. The sites of tagging correspond to ribosome stalling at the termination codon and at rare AGG codons encoding Arg-307 and Arg-309, the antepenultimate and C-terminal residues of
E. coli
ribokinase. Mutational analyses and studies of the effects of overexpressing the tRNA that decodes AGG reveal that the combination of a rare arginine codon at the C terminus and the adjacent inefficient UGA termination codon act to recruit the SsrA-tagging system, presumably by slowing the rate of translation elongation and termination.
Synaptic and extrasynaptic γ-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development
Authors: Scotti Alessandra L. , Reuter Harald
Published: July 2002

We have simultaneously measured the expression of postsynaptic
γ-aminobutyric acid type A (GABA
A
) receptor clusters and
of presynaptic boutons in neonatal rat hippocampal cultures between
days 1 and 30. GABA
A
receptors were labeled with antibodies
recognizing the extracellular domains of β2/3 and γ2
subunits. Boutons were visualized by activity-dependent uptake of the
styryl dye FM4-64, or by antibodies against the presynaptic vesicular
protein SV2 or the GABA-synthesizing enzyme glutamic acid decarboxylase
(GAD). GABA
A
receptor clusters could be seen in living
neurons already 6 h after culturing, much before presynaptic
markers could be identified in nerve terminals. The densities of
receptor clusters that contained the β2/3 subunits were
constant between days 10 and 30 in culture, whereas γ2
subunit-containing clusters fluctuated and reached a maximum on day 20.
SV2 and GAD staining could be measured from day 2 onwards. Clustering
of GAD in presynaptic terminals and FM4-64 uptake were observed only at
day 5 and afterward. SV2 staining and FM4-64 uptake increased in
parallel between days 5 and 20 and remained constant thereafter.
GAD-stained boutons were fewer than those labeled with other, less
specific, presynaptic stains. They reached a maximum on day 20 and fell
again toward day 30. Double labeling of GABA
A
receptors and
of presynaptic boutons in neurons during differentiation showed that,
even after 30 days in culture, large fractions of GABA
A
receptor clusters containing β2/3 and/or γ2 subunits
remained extrasynaptic.
Purification and characterization of an autoregulatory substance capable of regulating the morphological transition in
Candida albicans
Authors: Oh Ki-Bong , Miyazawa Hiroshi , Naito Toshimichi , Matsuoka Hideaki
Published: July 2002

The yeast
Candida albicans
has a distinguishing
feature, dimorphism, which is the ability to switch between two
morphological forms: a budding yeast form and a multicellular invasive
filamentous form. This ability has been postulated to contribute to the
virulence of this organism. Studies on the morphological transition
from a filamentous to a budding yeast form in
C.
albicans
have shown that this organism excretes an
autoregulatory substance into the culture medium. This substance was
extracted and purified by normal-phase and reversed-phase HPLC. The
autoregulatory substance was structurally identified as
3,7,11-trimethyl-2,6,10-dodecatrienoate (farnesoic acid) by NMR and
mass spectrometry. Growth experiments suggest that this substance does
not inhibit yeast cell growth but inhibits filamentous growth. These
findings have implications for developmental signaling by the fungus
and might have medicinal value in the development of antifungal
therapies.
Desensitization of μ-opioid receptor-evoked potassium currents: Initiation at the receptor, expression at the effector
Authors: Blanchet Christophe , Lüscher Christian
Published: July 2002

Many G protein-coupled receptor-mediated responses desensitize within minutes. Sustained stimulation of μ-opioid receptors (MORs), which primarily signal through G
i/o
proteins, leads to activation and subsequent desensitization of G protein-coupled inwardly rectifying potassium (GIRK) currents. We observed that in neurons of the locus coeruleus, which express among the highest levels of MORs in the brain, the degree of desensitization depended on the intensity of receptor stimulation, indicating that the process is initiated at the receptor. Interestingly, while GIRK-mediated postsynaptic inhibition substantially desensitized within 15 min, presynaptic inhibition of afferent transmission, which involves other effector systems, remained constant, suggesting that the postsynaptic desensitization we observed is expressed at the effector. We show that desensitized GIRK currents can gradually be reactivated by additional G protein signals of increasing intensity and present evidence that desensitization is a G protein-mediated process. Finally, desensitization of MOR-induced GIRK currents had heterologous effects on responses mediated by other G protein-coupled receptors converging onto the same population of GIRK channels. Taken together, our results provide evidence for a form of desensitization mediated by a slowly developing G protein-dependent pathway, initiated at the MORs and leading to competitive inhibition of GIRK channel activation. This implies that MORs exert a bidirectional action on GIRK channels.
Extending supramolecular fullerene-porphyrin chemistry to pillared metal-organic frameworks
Authors: Sun Dayong , Tham Fook S. , Reed Christopher A. , Boyd Peter D. W.
Published: July 2002

Porphyrins and fullerenes are spontaneously attracted to each other. This supramolecular recognition element can be exploited to produce ordered arrays of interleaved porphyrins and fullerenes. C60⋅H2TpyP⋅Pb(NO3)2⋅1.5TCE (H2TpyP = tetra-4-pyridylporphyrin; TCE = 1,1,2,2-tetrachloroethane) crystallizes in the tetragonalP4/nspace group and the structure has been solved to high resolution. The Pb2+ions connect the pyridylporphyrins in infinite sheets with an interlayer spacing of 12.1 Å. The fullerenes are intercalated between these layers, acting as pillars. The 6:6 ring juncture bonds of C60are centered over the porphyrins, bringing the layers into strict tetragonal register. This arranagement identifies the fullerene–porphyrin interaction as a structure-defining element. The same motif is seen in a related ribbon structure having C70intercalated into HgI2-linked H2TpyTP. The supramolecular design principles involved in assembling these chromophores may have applications in materials science.
Gramicidin A channels switch between stretch activation and stretch inactivation depending on bilayer thickness
Authors: Martinac Boris , Hamill Owen P.
Published: July 2002

The patch clamp-liposome technique was used to examine the stretch sensitivity of a model membrane ion channel, gramicidin A, in membrane patches of different bilayer thickness. We found that small changes in phospholipid acyl chain length (i.e., PC-20 to PC-18) can switch gramicidin A from a stretch-activated to a stretch-inactivated channel. The demonstration that subnanometer changes in bilayer thickness can reverse the response polarity of a model channel has implications for other signaling proteins that may experience local changes in bilayer thickness as a consequence of dynamic targeting to lipid microdomains, electrocompression, or chemical modification of the bilayer.
A chloroplast-resident DNA methyltransferase is responsible for hypermethylation of chloroplast genes in
Chlamydomonas
maternal gametes
Authors: Nishiyama Rie , Ito Mikako , Yamaguchi Yube , Koizumi Nozomu , Sano Hiroshi
Published: July 2002

Chloroplast DNA of the green alga
Chlamydomonas reinhardtii
is maternally inherited. Methylation mapping directly revealed that, before mating, chloroplast DNA of maternal (mating type plus; mt
+
) gametes is heavily methylated whereas that of paternal (mating type minus; mt
−
) gametes is not. Indirect immunofluorescence analyses with anti-5-methylcytosine mAbs visually showed methylation to occur exclusively in chloroplast DNA of mt
+
gametes, and not in mt
−
gametes or nuclear DNA of either mt. To clarify the relationship between methylation and maternal inheritance of chloroplast DNA, we have isolated and characterized a cDNA encoding a DNA methyltransferase. The deduced protein, CrMET1, consists of 1,344 aa and contains a conserved catalytic domain at the C terminal and a nonconserved N-terminal region. The predicted N-terminal region has an arginine-rich domain, suggesting CrMET1 is transferred to chloroplasts. This finding could be directly shown by green fluorescent protein epifluorescence microscopy analyses.
CrMET1
transcripts were found to be absent in both mt
+
and mt
−
vegetative cells. Upon gametogenesis, however, transcript levels clearly increased in mt
+
but not mt
−
cells. These experiments suggest that the CrMET1 protein is located in chloroplasts and that it specifically methylates cytosine residues of chloroplast DNA in mt
+
gametes. This conclusion was further strengthened by the observation that, during gametogenesis,
CrMET1
is expressed in a mt
−
mutant,
mat-1
, whose chloroplast DNA is heavily methylated in gametes and paternally inherited. The results provide evidence that cytosine methylation plays a critical role in maternal inheritance of chloroplast genes in
C. reinhardtii
.
Structure theorems for game trees
Authors: Govindan Srihari , Wilson Robert
Published: July 2002

Kohlberg and Mertens [Kohlberg, E. & Mertens, J. (1986)
Econometrica
54, 1003–1039] proved that the graph of the Nash equilibrium correspondence is homeomorphic to its domain when the domain is the space of payoffs in normal-form games. A counterexample disproves the analog for the equilibrium outcome correspondence over the space of payoffs in extensive-form games, but we prove an analog when the space of behavior strategies is perturbed so that every path in the game tree has nonzero probability. Without such perturbations, the graph is the closure of the union of a finite collection of its subsets, each diffeomorphic to a corresponding path-connected open subset of the space of payoffs. As an application, we construct an algorithm for computing equilibria of an extensive-form game with a perturbed strategy space, and thus approximate equilibria of the unperturbed game.
γ-Aminobutyric acid type A receptors modulate cAMP-mediated long-term potentiation and long-term depression at monosynaptic CA3–CA1 synapses
Authors: Yu Tzu-ping , McKinney Sheri , Lester Henry A. , Davidson Norman
Published: July 2002

cAMP induces a protein-synthesis-dependent late phase of long-term
potentiation (LTP) at CA3–CA1 synapses in acute hippocampal slices.
Herein we report cAMP-mediated LTP and long-term depression (LTD)
at monosynaptic CA3–CA1 cell pairs in organotypic hippocampal
slice cultures. After bath application of the membrane-permeable cAMP
analog adenosine 3′,5′-cyclic monophosphorothioate, Sp isomer
(Sp-cAMPS), synaptic transmission was enhanced for at least 2 h.
Consistent with previous findings, the late phase of LTP requires
activation of cAMP-dependent protein kinase A and protein synthesis.
There is also an early phase of LTP induced by cAMP; the early phase
depends on protein kinase A but, in contrast to the later phase, does
not require protein synthesis. In addition, the cAMP-induced LTP is
associated with a reduction of paired-pulse facilitation, suggesting
that presynaptic modification may be involved. Furthermore, we found
that Sp-cAMPS induced LTD in slices pretreated with picrotoxin, a
γ-aminobutyric acid type A (GABA
A
) receptor antagonist.
This form of LTD depends on protein synthesis and protein
phosphatase(s) and is accompanied by an increased ratio of failed
synaptic transmission. These results suggest that GABA
A
receptors can modulate the effect of cAMP on synaptic transmission and
thus determine the direction of synaptic plasticity.
Changes in global gene expression patterns during development and maturation of the rat kidney
Authors: Stuart Robert O. , Bush Kevin T. , Nigam Sanjay K.
Published: July 2002

We set out to define patterns of gene expression during kidney
organogenesis by using high-density DNA array technology. Expression
analysis of 8,740 rat genes revealed five discrete patterns or groups
of gene expression during nephrogenesis. Group 1 consisted of genes
with very high expression in the early embryonic kidney, many with
roles in protein translation and DNA replication. Group 2 consisted of
genes that peaked in midembryogenesis and contained many transcripts
specifying proteins of the extracellular matrix. Many additional
transcripts allied with groups 1 and 2 had known or proposed roles in
kidney development and included LIM1, POD1, GFRA1, WT1, BCL2, Homeobox
protein A11, timeless, pleiotrophin, HGF, HNF3, BMP4, TGF-α,
TGF-β2, IGF-II, met, FGF7, BMP4, and ganglioside-GD3. Group 3
consisted of transcripts that peaked in the neonatal period and
contained a number of retrotransposon RNAs. Group 4 contained genes
that steadily increased in relative expression levels throughout
development, including many genes involved in energy metabolism and
transport. Group 5 consisted of genes with relatively low levels of
expression throughout embryogenesis but with markedly higher levels in
the adult kidney; this group included a heterogeneous mix of
transporters, detoxification enzymes, and oxidative stress genes. The
data suggest that the embryonic kidney is committed to cellular
proliferation and morphogenesis early on, followed sequentially by
extracellular matrix deposition and acquisition of markers of terminal
differentiation. The neonatal burst of retrotransposon mRNA was
unexpected and may play a role in a stress response associated with
birth. Custom analytical tools were developed including “The
Equalizer” and “eBlot,” which contain improved methods for
data normalization, significance testing, and data mining.
IRF-3-dependent, NFκB- and JNK-independent activation of the 561 and IFN-β genes in response to double-stranded RNA
Authors: Peters Kristi L. , Smith Heather L. , Stark George R. , Sen Ganes C.
Published: July 2002

Double-stranded (ds) RNA induces transcription of the 561 gene by activating IFN regulatory factor (IRF) transcription factors, whereas similar induction of the IFN-β gene is thought to require additional activation of NFκB and AP-1. In mutant P2.1 cells, dsRNA failed to activate NFκB, IRF-3, p38, or c-Jun N-terminal kinase, and transcription of neither 561 mRNA nor IFN-β mRNA was induced. The defect in the IRF-3 pathway was traced to a low cellular level of this protein because of its higher rate of degradation in P2.1 cells. As anticipated, in several clonal derivatives of P2.1 cells expressing different levels of transfected IRF-3, activation of IRF-3 and induction of 561 mRNA by dsRNA was restored fully, although the defects in other responses to dsRNA persisted. Surprisingly, IFN-β mRNA also was induced strongly in these cells in response to dsRNA, demonstrating that the activation of NFκB and AP-1 is not required. This conclusion was confirmed in wild-type cells overexpressing IRF-3 by blocking NFκB activation with the IκB superrepressor and AP-1 activation with a p38 inhibitor. Therefore, IRF-3 activation by dsRNA is sufficient to induce the transcription of genes with simple promoters such as 561 as well as complex promoters such as IFN-β.
Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils
Authors: DeCoursey T. E. , Cherny V. V. , Zhou W. , Thomas L. L.
Published: July 2002

Generation of reactive oxygen species by the NADPH oxidase complex is an important bactericidal weapon of phagocytes. Phorbol myristate acetate (PMA) is a potent agonist for this “respiratory burst” in human neutrophils. Although stoichiometric H
+
efflux occurs during the respiratory burst, efforts to stimulate voltage-gated H
+
channels by PMA in whole-cell patch-clamped phagocytes have been unsuccessful. We have used a modification of the permeabilized-patch configuration that allows control of intracellular pH and preserves second-messenger pathways. Using this method, we show that PMA dramatically enhances and alters voltage-gated proton currents in human neutrophils. PMA produced four alterations in H
+
current properties, each of which increases the H
+
current at any given voltage: (
i
) a 40-mV negative shift in the H
+
conductance-voltage (
g
H
–
V
) relationship; (
ii
) faster activation [smaller activation time constant (τ
act
)] during depolarizing pulses; (
iii
) slower deactivation [larger deactivation time constant (τ
tail
)] on repolarization; and (
iv
) a larger maximum H
+
conductance (
g
H,max
). Inward current that directly reflects electron transport by NADPH oxidase was also activated by PMA stimulation. The identity of this electron current was confirmed by its sensitivity to diphenylene iodinium, an inhibitor of NADPH oxidase. Diphenylene iodinium also reversed the slowing of τ
tail
with a time course paralleling the inhibition of electron current. However, the amplitudes of H
+
and electron currents activated by PMA were not correlated. A complex interaction between NADPH oxidase and voltage-gated proton channels is indicated. The data suggest that PMA stimulation modulates preexisting H
+
channels rather than inducing a new H
+
channel.
Elevated mutation rates in the germ line of first- and second-generation offspring of irradiated male mice
Authors: Barber Ruth , Plumb Mark A. , Boulton Emma , Roux Isabelle , Dubrova Yuri E.
Published: July 2002

Mutation rates at two expanded simple tandem repeat loci were studied in the germ line of first- and second-generation offspring of inbred male CBA/H, C57BL/6, and BALB/c mice exposed to either high linear energy transfer fission neutrons or low linear energy transfer x-rays. Paternal CBA/H exposure to either x-rays or fission neutrons resulted in increased mutation rates in the germ line of two subsequent generations. Comparable transgenerational effects were observed also in neutron-irradiated C57BL/6 and x-irradiated BALB/c mice. The levels of spontaneous mutation rates and radiation-induced transgenerational instability varied between strains (BALB/c>CBA/H>C57BL/6). Pre- and postmeiotic paternal exposure resulted in similar increases in mutation rate in the germ line of both generations of CBA/H mice, which together with our previous results suggests that radiation-induced expanded simple tandem repeat instability is manifested in diploid cells after fertilization. The remarkable finding that radiation-induced germ-line instability persists for at least two generations raises important issues of risk evaluation in humans.
Opposing actions of adrenal androgens and glucocorticoids on alternative splicing of
Slo
potassium channels in bovine chromaffin cells
Authors: Lai Guey-Jen , McCobb David P.
Published: July 2002

Pituitary ablation (hypophysectomy) in rats was previously reported to cause a precipitous change in the relative abundance of two alternative splice variants of the “BK”- or “Maxi K”-encoding
Slo
gene in adrenal chromaffin cells. Inclusion of the optional “STREX” exon (STRess axis-regulated EXon) in a C-terminal splice site was reduced, in preference to the variant lacking an insert at this site. Adrenocorticotropic hormone (ACTH) injections prevented the drop in STREX inclusion, implicating stress-axis function, as opposed to other pituitary functions. Because ACTH promotes synthesis and release of glucocorticoids (corticosterone or cortisol, depending on species), we hypothesized that glucocorticoids applied directly would promote STREX inclusion. Contrary to predictions, we report that direct application of glucocorticoids to bovine cells
in vitro
decreased STREX inclusion. This effect was blocked by the glucocorticoid receptor antagonist RU38486. As with glucocorticoids, synthesis and release of the adrenal androgen dehydroepiandrosterone (DHEA) increases in response to stress or elevated ACTH levels in some species. We report that direct application of DHEA increased expression of the STREX variant in cultured bovine cells. Two other androgens, androstenedione and testosterone, had similar effects. We hypothesize that Slo splicing in adrenal chromaffin cells
in vivo
is differentially regulated by the integrative, concentration- and time-dependent actions of glucocorticoids and androgens, with potentially important ramifications for stress-evoked catecholamine secretion.
Atomic force microscopy reveals the mechanical design of a modular protein
Authors: Li Hongbin , Oberhauser Andres F. , Fowler Susan B. , Clarke Jane , Fernandez Julio M.
Published: July 2002

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.
A yeast gene,
MGS1
, encoding a DNA-dependent AAA
+
ATPase is required to maintain genome stability
Authors: Hishida Takashi , Iwasaki Hiroshi , Ohno Takayuki , Morishita Takashi , Shinagawa Hideo
Published: July 2002

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a
Saccharomyces cerevisiae
gene,
MGS1
(
M
aintenance of
G
enome
S
tability 1), which encodes a protein belonging to the AAA
+
class of ATPases, and whose central region is similar to
Escherichia coli
RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An
mgs1
deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The
mgs1
mutation is synergistic with a mutation in
top3
(encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the
mgs1
mutation, a mutation in the
sgs1
gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the
top3
mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.
Crystal structure of the SarR protein from
Staphylococcus aureus
Authors: Liu Yingfang , Manna Adhar , Li Ronggui , Martin Wesley E. , Murphy Robert C. , Cheung Ambrose L. , Zhang Gongyi
Published: July 2002

The expression of virulence determinants in
Staphylococcus
aureus
is controlled by global regulatory loci (e.g.,
sarA
and
agr
). The
sar
(
Staphylococcus
accessory regulator) locus is composed
of three overlapping transcripts (
sarA
P1, P3, and P2,
transcripts initiated from the P1, P3, and P2 promoters, respectively),
all encoding the 124-aa SarA protein. The level of SarA, the major
regulatory protein, is partially controlled by the differential
activation of the
sarA
promoters. We previously
partially purified a 13.6-kDa protein, designated SarR, that binds to
the
sarA
promoter region to down-modulate
sar
A transcription from the P1 promoter and subsequently
SarA expression. SarR shares sequence similarity to SarA, and another
SarA homolog, SarS. Here we report the 2.3 Å-resolution x-ray crystal
structure of the dimeric SarR-MBP (maltose binding protein) fusion
protein. The structure reveals that the SarR protein not only has a
classic helix–turn–helix module for DNA binding at the major grooves,
but also has an additional loop region involved in DNA recognition at
the minor grooves. This interaction mode could represent a new
functional class of the “winged helix” family. The dimeric SarR
structure could accommodate an unusually long stretch of ≈27
nucleotides with two or four bending points along the course, which
could lead to the bending of DNA by 90° or more, similar to that seen
in the catabolite activator protein (CAP)–DNA complex. The structure
also demonstrates the molecular basis for the stable dimerization of
the SarR monomers and possible motifs for interaction with other
proteins.
Protein-coding genes are epigenetically regulated in
Arabidopsis
polyploids
Authors: Lee Hyeon-Se , Chen Z. Jeffrey
Published: July 2002

The fate of redundant genes resulting from genome duplication is
poorly understood. Previous studies indicated that ribosomal RNA genes
from one parental origin are epigenetically silenced during
interspecific hybridization or polyploidization. Regulatory mechanisms
for protein-coding genes in polyploid genomes are unknown, partly
because of difficulty in studying expression patterns of homologous
genes. Here we apply amplified fragment length polymorphism
(AFLP)–cDNA display to perform a genome-wide screen for orthologous
genes silenced in
Arabidopsis suecica
, an allotetraploid
derived from
Arabidopsis thaliana
and
Cardaminopsis arenosa
. We identified ten genes that are
silenced from either
A. thaliana
or
C.
arenosa
origin in
A. suecica
and located in four
of the five
A. thaliana
chromosomes. These genes
represent a variety of RNA and predicted proteins including four
transcription factors such as TCP3. The silenced genes in the vicinity
of
TCP3
are hypermethylated and reactivated by blocking
DNA methylation, suggesting epigenetic regulation is involved in the
expression of orthologous genes in polyploid genomes. Compared with
classic genetic mutations, epigenetic regulation may be advantageous
for selection and adaptation of polyploid species during evolution and
development.
Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation
Authors: Stenina Olga I. , Shaneyfelt Kristin M. , DiCorleto Paul E.
Published: July 2002

We have recently demonstrated that thrombin induces expression of the platelet-derived growth factor B-chain gene in endothelial cells (EC) through activation of the Y-box binding protein DNA-binding protein B (dbpB). We now present evidence that dbpB is activated by a novel mechanism: proteolytic cleavage leading to release from mRNA, nuclear translocation, and induction of thrombin-responsive genes. Cytosolic, full-length dbpB (50 kDa) was rapidly cleaved to a 30-kDa species upon thrombin stimulation of EC. This truncated, “active” dbpB exhibited nuclear localization and binding affinity for the thrombin response element sequence, which is distinct from the Y-box sequence. Oligo(dT) affinity chromatography revealed that cytosolic dbpB from control EC, but not active dbpB from thrombin-treated EC, was bound to mRNA. Latent dbpB immunoprecipitated from cytosolic extracts of control EC was activated by ribonuclease treatment. Furthermore, when EC cytosolic extracts were subjected to Nycodenz gradient centrifugation, latent dbpB fractionated with mRNA, whereas active dbpB fractionated with free proteins. The cytosolic retention domain of dbpB, which we localized to the region 247–267, was proteolytically cleaved during its activation. In contrast to full-length dbpB, truncated dbpB stimulated platelet-derived growth factor B-chain and tissue factor promoter activity by over 5-fold when transiently cotransfected with reporter constructs. These results suggest a novel mode of transcription factor activation in which an agonist causes release from mRNA of a latent transcription factor leading to its transport to the nucleus and its regulation of target gene expression.
Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX
Authors: Bassing Craig H. , Chua Katrin F. , Sekiguchi JoAnn , Suh Heikyung , Whitlow Scott R. , Fleming James C. , Monroe Brianna C. , Ciccone David N. , Yan Catherine , Vlasakova Katerina , Livingston David M. , Ferguson David O. , Scully Ralph , Alt Frederick W.
Published: July 2002

In mammalian cells, DNA double-strand breaks (DSBs) cause rapid phosphorylation of the H2AX core histone variant (to form γ-H2AX) in megabase chromatin domains flanking sites of DNA damage. To investigate the role of H2AX in mammalian cells, we generated H2AX-deficient (H2AX
Δ
/
Δ
) mouse embryonic stem (ES) cells. H2AX
Δ
/
Δ
ES cells are viable. However, they are highly sensitive to ionizing radiation (IR) and exhibit elevated levels of spontaneous and IR-induced genomic instability. Notably, H2AX is not required for NHEJ
per se
because H2AX
Δ
/
Δ
ES cells support normal levels and fidelity of V(D)J recombination in transient assays and also support lymphocyte development
in vivo
. However, H2AX
Δ
/
Δ
ES cells exhibit altered IR-induced BRCA1 focus formation. Our findings indicate that H2AX function is essential for mammalian DNA repair and genomic stability.
Interaction of the iron–sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of
Escherichia
coli
Authors: Hoff Kevin G. , Silberg Jonathan J. , Vickery Larry E.
Published: July 2002

The
iscU
gene in bacteria is located in a gene cluster encoding proteins implicated in
i
ron–
s
ulfur
c
luster assembly and an hsc70-type (
h
eat
s
hock
c
ognate) molecular chaperone system,
iscSUA-hscBA
. To investigate possible interactions between these systems, we have overproduced and purified the IscU protein from
Escherichia coli
and have studied its interactions with the
hscA
and
hscB
gene products Hsc66 and Hsc20. IscU and its iron–sulfur complex (IscU–Fe/S) stimulated the basal steady-state ATPase activity of Hsc66 weakly in the absence of Hsc20 but, in the presence of Hsc20, increased the ATPase activity up to 480-fold. Hsc20 also decreased the apparent
K
m
for IscU stimulation of Hsc66 ATPase activity, and surface plasmon resonance studies revealed that Hsc20 enhances binding of IscU to Hsc66. Surface plasmon resonance and isothermal titration calorimetry further showed that IscU and Hsc20 form a complex, and Hsc20 may thereby aid in the targeting of IscU to Hsc66. These results establish a direct and specific role for the Hsc66/Hsc20 chaperone system in functioning with
isc
gene components for the assembly of iron–sulfur cluster proteins.
Nanoparticle halos: A new colloid stabilization mechanism
Authors: Tohver Valeria , Smay James E. , Braem Alan , Braun Paul V. , Lewis Jennifer A.
Published: July 2002

A new mechanism for regulating the stability of colloidal particles has been discovered. Negligibly charged colloidal microspheres, which flocculate when suspended alone in aqueous solution, undergo a remarkable stabilizing transition upon the addition of a critical volume fraction of highly charged nanoparticle species. Zeta potential analysis revealed that these microspheres exhibited an effective charge buildup in the presence of such species. Scanning angle reflectometry measurements indicated, however, that these nanoparticle species did not adsorb on the microspheres under the experimental conditions of interest. It is therefore proposed that highly charged nanoparticles segregate to regions near negligibly charged microspheres because of their repulsive Coulombic interactions in solution. This type of nanoparticle haloing provides a previously unreported method for tailoring the behavior of complex fluids.
Learning about pain: The neural substrate of the prediction error for aversive events
Authors: Ploghaus Alexander , Tracey Irene , Clare Stuart , Gati Joseph S. , Rawlins J. Nicholas P. , Matthews Paul M.
Published: July 2002

Associative learning is thought to depend on detecting mismatches between actual and expected experiences. With functional magnetic resonance imaging (FMRI), we studied brain activity during different types of mismatch in a paradigm where contrasting-colored lights signaled the delivery of painful heat, nonpainful warmth, or no stimulation. When painful heat stimulation was unexpected, there was increased FMRI signal intensity in areas of the hippocampus, superior frontal gyrus, cerebellum, and superior parietal gyrus that was not found with mismatch between expectation and delivery of nonpainful warmth stimulation. When painful heat stimulation was unexpectedly omitted, the FMRI signal intensity decreased in the left superior parietal gyrus and increased in the other regions. These contrasting activation patterns correspond to two different mismatch concepts in theories of associative learning (Rescorla-Wagner, temporal difference vs. Pearce-Hall, Mackintosh). Searching for interventions to specifically modulate activation of these brain regions therefore offers an approach to identifying new treatments for chronic pain, which often has a substantial associative learning component.
In vivo
transposon mutagenesis of the methanogenic archaeon
Methanosarcina acetivorans
C2A using a modified version of the insect
mariner
-family transposable element
Himar1
Authors: Zhang J. K. , Pritchett M. A. , Lampe D. J. , Robertson H. M. , Metcalf W. W.
Published: July 2002

We present here a method for
in vivo
transposon mutagenesis of a methanogenic archaeon,
Methanosarcina acetivorans
C2A, which because of its independence from host-specific factors may have broad application among many microorganisms. Because there are no known
Methanosarcina
transposons we modified the
mariner
transposable element
Himar1,
originally found in the insect
Hematobia irritans
, to allow its use in this organism. This element was chosen because, like other
mariner
elements, its transposition is independent of host factors, requiring only its cognate transposase. Modified mini-
Himar1
elements were constructed that carry selectable markers that are functional in
Methanosarcina
species and that express the
Himar1
transposase from known
Methanosarcina
promoters. These mini-
mariner
elements transpose at high frequency in
M. acetivorans
to random sites in the genome. The presence of an
Escherichia coli
selectable marker and plasmid origin of replication within the mini-
mariner
elements allows facile cloning of these transposon insertions to identify the mutated gene. In preliminary experiments, we have isolated numerous mini-
mariner
-induced
M. acetivorans
mutants, including ones with insertions that confer resistance to toxic analogs and in genes that encode proteins involved in heat shock, nitrogen fixation, and cell-wall structures.
Interactions of Exo1p with components of MutLα in
Saccharomyces cerevisiae
Authors: Tran Phuoc T. , Simon Jeffrey A. , Liskay R. Michael
Published: July 2002

Previously, we reported evidence suggesting that
Saccharomyces cerevisiae
MutLα, composed of Mlh1p and
Pms1p, was a functional member of the gyrase b/Hsp90/MutL (GHL)
dimeric ATPase superfamily characterized by highly conserved ATPase
domains. Similar to other GHL ATPases, these putative ATPase domains of
MutLα may be important for the recruitment and/or activation of
downstream effectors. One downstream effector candidate is Exo1p, a
5′-3′ double stranded DNA exonuclease that has previously been
implicated in DNA mismatch repair (MMR). Here we report yeast
two-hybrid results suggesting that Exo1p can interact physically with
MutLα through the Mlh1p subunit. We also report epistasis analysis
involving MutLα ATPase mutations combined with
exo1
Δ. One interpretation of our genetic results is
that MutLα ATPase domains function to direct Exo1p and other
functionally redundant exonucleases during MMR. Finally, our results
show that much of the increase in spontaneous mutation observed in an
exo1
Δ strain is
REV3
-dependent, in turn
suggesting that Exo1p is also involved in one or more MMR-independent
mutation avoidance pathways.