bluuebunny/crossref_metadata_2025 huggingface dataset:
Noninvasive sheet charge density probe for integrated silicon devices
Authors: Heinrich H. K. , Bloom D. M. , Hemenway B. R.
Published: July 2002

We report a sensitive new technique for probing dynamic sheet charge density variations in integrated silicon devices. Using a specially designed noninvasive Nomarski phase contrast interferometer a sheet charge density sensitivity of 2.6×108 e/cm2/(Hz)1/2 is extracted from experimental data for 1 mA of detected photocurrent. This charge density sensitivity makes possible μV signal level detection in an active device, and with digital signals the corresponding signal/noise level is sufficiently high that multimegabaud data can be captured in real time.
Room-temperature cw operation of InGaP/InGaAlP visible light laser diodes on GaAs substrates grown by metalorganic chemical vapor deposition
Authors: Ishikawa Masayuki , Ohba Yasuo , Sugawara Hideto , Yamamoto Motoyuki , Nakanisi Takatosi
Published: July 2002

Room-temperature cw operation for InGaP/InGaAlP double heterostructure (DH) laser diodes on GaAs substrates was achieved for the first time. The DH wafers were grown by low-pressure metalorganic chemical vapor deposition using methyl metalorganics. A lasing wavelength of 679 nm and a threshold current of 109 mA at 24 °C were obtained for an inner stripe structure laser diode with a 250-μm-long and 7-μm stripe geometry. The laser operated at up to 51 °C. The characteristic temperature T0 was 87 K at around room temperature. The lowest threshold current density, 5.0 kA/cm2, was obtained with a 20-μm stripe width laser diode under room-temperature pulsed operation.
Observation of enhanced single longitudinal mode operation in 1.5-μm GaInAsP erbium-doped semiconductor injection lasers
Authors: Tsang W. T. , Logan R. A.
Published: July 2002

We propose a new type of current injection semiconductor laser with rare-earth dopant in the active layer for achieving stable, single longitudinal mode operation. In this laser, the rare-earth/semiconductor combination is chosen such that the wavelengths of the dominant emission from the trivalent rare-earth ion internal 4f-4f  transitions are shorter than that of the band-edge emission of the host semiconductor. Spectrally, the narrow optical gain due to the rare-earth ion transition will superimpose on top of the broad gain peak of the host semiconductor. Such laser diodes will attain lasing action at the rare-earth transition wavelength resulting in single longitudinal mode operation with conventional Fabry–Perot cavity. Furthermore, reproducible precise lasing wavelength insensitive to temperature variation should be possible. Such a proposed rare-earth/semiconductor injection laser was investigated with erbium doping in the GaInAsP (λ=1.55 μm) active layer of the heteroepitaxial ridge-overgrown laser. Stable, single longitudinal mode operation at 15 322 Å was obtained. Furthermore, this lasing line shifted at a slow rate of ≲1 Å/°C with heat-sink temperature. Initial results do appear to confirm this new proposed rare-earth/semiconductor laser operation scheme.
Charge transfer enhancement of photoconductivity in hydrogenated amorphous Ge/Si multilayer films
Authors: Wronski C. R. , Tiedje T. , Persans P. , Abeles B. , Hicks M.
Published: July 2002

Enhancement of amorphous Ge photoconductivity is observed in hydrogenated amorphous Ge/Si (a-Ge:H/a-Si:H) multilayer structures. The increase in electron lifetimes is explained in terms of a novel sensitization effect due to transfer of trapped holes from the a-Ge:H to the a-Si:H layers.
Mapping of microelectric and magnetic fields with double-exposure electron holography
Authors: Matteucci G. , Missiroli G. F. , Chen J. W. , Pozzi G.
Published: July 2002

The double-exposure electron holographic technique has been put into practical use for the first time. By this method an accurate recording of the distribution of electric and magnetic fields can be directly obtained by the electron microscope without resorting to sophisticated optical manipulation of the holograms. Problems concerning the operative definition of the contour maps are discussed. Experimental results are presented.
Nuts, nut cracking, and pitted stones at Gesher Benot Ya‘aqov, Israel
Authors: Goren-Inbar Naama , Sharon Gonen , Melamed Yoel , Kislev Mordechai
Published: July 2002

The Acheulian site of Gesher Benot Ya‘aqov (Israel) has revealed a unique association of edible nuts with pitted hammers and anvils. Located in the Dead Sea rift, on the boundary between the Arabian and African plates, the site dates to the Early-Middle Pleistocene, oxygen isotope stage 19. In a series of strata, seven species of nuts, most of which can be cracked open only by a hard hammer, were uncovered. Five of the species are extant terrestrial nuts, and two are aquatic nuts now extinct in the Levant. In addition, the site yielded an assemblage of pitted hammers and anvils similar in pit morphology to those used by chimpanzees and contemporary hunter–gatherers. This is the first time, to our knowledge, that a site has offered both paleobotanical and lithic evidence of plant foods eaten by early hominins and technologies used for processing these foods. The evidence also sheds light on the structure of the community: ethnographic analogies suggest that mixedgender groups may have been active on the shores of paleoLake Hula.
Probing protein conformational changes in living cells by using designer binding proteins: Application to the estrogen receptor
Authors: Koide Akiko , Abbatiello Stacy , Rothgery Lisa , Koide Shohei
Published: July 2002

A challenge in understanding the mechanism of protein function in biology is to establish the correlation between functional form in the intracellular environment and high-resolution structures obtained with
in vitro
techniques. Here we present a strategy to probe conformational changes of proteins inside cells. Our method involves: (
i
) engineering binding proteins to different conformations of a target protein, and (
ii
) using them to sense changes in the surface property of the target in cells. We probed ligand-induced conformational changes of the estrogen receptor α (ERα) ligand-binding domain (LBD). By using yeast two-hybrid techniques, we first performed combinatorial library screening of “monobodies” (small antibody mimics using the scaffold of a fibronectin type III domain) for clones that bind to ERα and then characterized their interactions with ERα in the nucleus, the native environment of ERα, in the presence of various ligands. A library using a highly flexible loop yielded monobodies that specifically recognize a particular ligand complex of ERα, and the pattern of monobody specificity was consistent with the structural differences found in known crystal structures of ERα-LBD. A more restrained loop library yielded clones that bind both agonist- and antagonist-bound ERα. Furthermore, we found that a deletion of the ERα F domain that is C-terminally adjacent to the LBD increased the crossreactivity of monobodies to the apo-ERα-LBD, suggesting a dynamic nature of the ERα-LBD conformation and a role of the F domain in restraining the LBD in an inactive conformation.
Widanelfarasia, a diminutive placental from the late Eocene of Egypt
Authors: Seiffert Erik R. , Simons Elwyn L.
Published: July 2002

The lower dentition ofWidanelfarasia(new genus), a diminutive late Eocene placental from the Fayum Depression in Egypt, is described.Widanelfarasiaexhibits a complex of features associated with incipient zalambdodonty and at least three unequivocal apomorphies [loss of P1, an enlarged I2(relative to I3), and a basal cusp on I2], which provide weak support for its placement as a possible sister taxon of either a tenrecid–chrysochlorid clade or of solenodontids. The former hypothesis gains additional support from biogeographical evidence, but both scenarios are currently tenuous asWidanelfarasiais clearly not truly zalambdodont. Phylogenetic hypotheses positing affinities with tenrecids alone or chrysochlorids alone must invoke either convergent acquisition of zalambdodonty in these taxa or autapomorphic reversal inWidanelfarasia. Given these considerations, a relationship with more generalized taxa from the Laurasian Paleogene (e.g., geolabidids, nyctitheriids, leptictids) cannot yet be ruled out. Comparisons with other Paleogene Afro-Arabian forms are generally inconclusive. A relationship with the earlier EoceneChambilestesfrom Tunisia—currently represented by a single specimen preserving P4–M3—seems possible based on the geometry and predicted occlusal relationships of these teeth, but cannot be confidently determined until these two taxa come to be represented by common diagnostic elements.Todralestes(late Paleocene, Morocco) exhibits general phenetic similarities toWidanelfarasia, but it is not yet known whether this taxon shares any ofWidanelfarasia's unequivocal dental apomorphies. Pending the recovery of more informative material, we tentatively referWidanelfarasiato Placentaliaincertae sedis. Truly zalambdodont placentals remain conspicuously absent from the Paleogene of Afro-Arabia.
Microbial phyllosphere populations are more complex than previously realized
Authors: Yang Ching-Hong , Crowley David E. , Borneman James , Keen Noel T.
Published: July 2002

Phyllosphere microbial communities were evaluated on leaves of
field-grown plant species by culture-dependent and -independent
methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA
primers generally indicated that microbial community structures were
similar on different individuals of the same plant species, but unique
on different plant species. Phyllosphere bacteria were identified from
Citrus sinesis
(cv. Valencia) by using DGGE analysis
followed by cloning and sequencing of the dominant rDNA bands. Of the
17 unique sequences obtained, database queries showed only four strains
that had been described previously as phyllosphere bacteria. Five of
the 17 sequences had 16S similarities lower than 90% to database
entries, suggesting that they represent previously undescribed species.
In addition, three fungal species were also identified. Very different
16S rDNA DGGE banding profiles were obtained when replicate cv.
Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days.
All of these rDNA sequences had 97–100% similarity to those of known
phyllosphere bacteria, but only two of them matched those identified by
the culture independent DGGE analysis. Like other studied ecosystems,
microbial phyllosphere communities therefore are more complex than
previously thought, based on conventional culture-based methods.
The tRNA Specificity of
Thermus thermophilus
EF-Tu
Authors: Asahara Haruichi , Uhlenbeck Olke C.
Published: July 2002

By introducing a GAC anticodon, 21 different
Escherichia coli
tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to
Thermus thermophilus
elongation factor Tu (EF-Tu)⋅GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The
E. coli
elongator tRNAs exhibit a wide range of binding affinities that varied from −11.7 kcal/mol for Val-tRNA
Glu
to −8.1 kcal/mol for Val-tRNA
Tyr
, clearly establishing EF-Tu⋅GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of
k
off
varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism.
Evidence for
in vivo
modulation of chloroplast RNA stability by 3′-UTR homopolymeric tails in
Chlamydomonas reinhardtii
Authors: Komine Yutaka , Kikis Elise , Schuster Gadi , Stern David
Published: July 2002

Polyadenylation of synthetic RNAs stimulates rapid degradation
in vitro
by using either
Chlamydomonas
or spinach chloroplast extracts. Here, we used
Chlamydomonas
chloroplast transformation to test the effects of mRNA homopolymer tails
in vivo
, with either the endogenous
atpB
gene or a version of green fluorescent protein developed for chloroplast expression as reporters. Strains were created in which, after transcription of
atpB
or
gfp
, RNase P cleavage occurred upstream of an ectopic tRNA
Glu
moiety, thereby exposing A
28
, U
25
A
3
, [A+U]
26
, or A
3
tails. Analysis of these strains showed that, as expected, polyadenylated transcripts failed to accumulate, with RNA being undetectable either by filter hybridization or reverse transcriptase–PCR. In accordance, neither the ATPase β-subunit nor green fluorescent protein could be detected. However, a U
25
A
3
tail also strongly reduced RNA accumulation relative to a control, whereas the [A+U] tail did not, which is suggestive of a degradation mechanism that does not specifically recognize poly(A), or that multiple mechanisms exist. With an A
3
tail, RNA levels decreased relative to a control with no added tail, but some RNA and protein accumulation was observed. We took advantage of the fact that the strain carrying a modified
atpB
gene producing an A
28
tail is an obligate heterotroph to obtain photoautotrophic revertants. Each revertant exhibited restored
atpB
mRNA accumulation and translation, and seemed to act by preventing poly(A) tail exposure. This suggests that the poly(A) tail is only recognized as an instability determinant when exposed at the 3′ end of a message.
Disrupting the
IL-4
gene rescues mice homozygous for the tight-skin mutation from embryonic death and diminishes TGF-β production by fibroblasts
Authors: Kodera Takao , McGaha Tracy L. , Phelps Robert , Paul William E. , Bona Constantin A.
Published: July 2002

The
TSK
/
TSK
mutation is embryonic lethal; embryos have been reported to die at 7–8 days of gestational age. Crossing
TSK
/+,
IL-4
+/− mice revealed that disrupting one or both
IL-4
alleles allowed survival of 29 and 47%, respectively, of
TSK
/
TSK
mice. These mice failed to develop cutaneous hyperplasia but did exhibit the emphysema that is found in
TSK
/+ mice. We showed that IL-4 stimulation of fibroblasts increased the level of transforming growth factor-β (TGF-β) mRNA and that lungs of
TSK
/+,
IL-4
−/− mice had substantially less TGF-β mRNA than lungs of
TSK
/+,
IL-4
+/+ mice. Thus IL-4 seems to regulate the expression of TGF-β in fibroblasts, providing an explanation for the absence of cutaneous hyperplasia in
TSK
/+,
IL-4R
α−/− and
TSK
/+,
TGF-
β+/− mice.
Conserved tryptophan in the core domain of transglutaminase is essential for catalytic activity
Authors: Murthy S. N. Prasanna , Iismaa Siiri , Begg Gillian , Freymann Douglas M. , Graham Robert M. , Lorand Laszlo
Published: July 2002

Transglutaminase 2 (TG2) is a distinctive member of the family of Ca
2+
-dependent enzymes recognized mostly by their abilities to catalyze the posttranslational crosslinking of proteins. TG2 uniquely binds and hydrolyzes GTP; binding GTP inhibits its crosslinking activity but allows it to function in signal transduction (hence the G
h
designation). The core domain of TG2 (residues 139–471, rat) comprises the papain-like catalytic triad and the GTP-binding domain (residues 159–173) and contains almost all of the conserved tryptophans of the protein. Examining point mutations at Trp positions 180, 241, 278, 332, and 337 showed that, upon binding 2′-(or 3′)-
O
-(
N
-methylanthraniloyl)GTP (mantGTP), the Phe-332 mutant was the weakest (35% less than wild type) in resonance energy transfer from the protein (λ
exc, max
= 290 nm) to the mant fluorophore (λ
em
= 444 nm) and had a reduced affinity for mantGTP. Trp-332, situated near the catalytic center and the nucleotide-binding area of TG2, may be part of the allosteric relay machinery that transmits negative effector signals from nucleotide binding to the active center of TG2. A most important observation was that, whereas no enzyme activity could be detected when Trp-241 was replaced with Ala or Gln, partial preservation of catalytic activity was seen with substitutions by Tyr > Phe > His. The results indicate that Trp-241 is essential for catalysis, possibly by stabilizing the transition states by H-bonding, quadrupole–ion, or van der Waals interactions. This contrasts with the evolutionarily related papain family of cysteine proteases, which uses Gln-19 (papain) for stabilizing the transition state.
GCR1
, the putative
Arabidopsis
G protein-coupled receptor gene is cell cycle-regulated, and its overexpression abolishes seed dormancy and shortens time to flowering
Authors: Colucci Gabriella , Apone Fabio , Alyeshmerni Nicole , Chalmers Derek , Chrispeels Maarten J.
Published: July 2002

Although signaling through heterotrimeric G proteins has been extensively studied in eukaryotes, there is little information about this important signaling pathway in plants. We observed that expression of
GCR1
, the gene encoding the only known (but still putative) G protein-coupled receptor of
Arabidopsis thaliana
, is modulated during the cell cycle and during plant development. Overexpression of
GCR1
in tobacco (
Nicotiana tabacum
) BY-2 cells caused an increase in thymidine incorporation and in the mitotic index of aphidicolin synchronized cells. Overexpression of
GCR1
in
Arabidopsis
caused two remarkable phenotypes: seed dormancy was abolished and time to flowering was reduced. Molecular markers of these two developmental processes (phosphatase
PP2A
and
MYB65
in germination;
LFY
during flowering) were up-regulated in
GCR1
overexpressors. These data are consistent with the hypothesis that
GCR1
may be a regulator of the cell cycle and that this regulation underlies the developmental changes observed in the
GCR1
transformants.
Coping with crowds: Density-dependent disease resistance in desert locusts
Authors: Wilson Kenneth , Thomas Matthew B. , Blanford Simon , Doggett Matthew , Simpson Stephen J. , Moore Sarah L.
Published: July 2002

Parasite transmission generally exhibits some form of positive density dependence. Thus, as population density increases, so too does theper capitarisk of becoming infected. Under such circumstances, natural selection should favor individuals that use cues associated with population density to determine the optimal allocation of resources to disease resistance mechanisms. As a consequence, individuals experiencing crowded conditions are predicted to be more resistant to parasites and pathogens than those experiencing low-density conditions. This phenomenon (termed “density-dependent prophylaxis”) [Wilson, K. & Reeson, A. F. (1998)Ecol. Entomol.23, 100–101] is predicted to be particularly prevalent in outbreak pest species and in species exhibiting density-dependent phase polyphenism, such as the desert locust,Schistocerca gregaria. Here we show that, as predicted, desert locusts reared under crowded conditions are significantly more resistant than solitary locusts to the entomopathogenic fungus,Metarhizium anisopliaevar.acridum, a key natural disease of acridids and an important agent in locust and grasshopper biocontrol. Moreover, enhanced pathogen resistance in crowded locusts is associated with elevated antimicrobial activity, but not with any difference in thermal preferences or behavioral fever response. These results have implications for understanding the development and biocontrol of locust plagues.
The evolution of coloration and toxicity in the poison frog family (Dendrobatidae)
Authors: Summers Kyle , Clough Mark E.
Published: July 2002

The poison frogs (family Dendrobatidae) are terrestrial anuran amphibians displaying a wide range of coloration and toxicity. These frogs generally have been considered to be aposematic, but relatively little research has been carried out to test the predictions of this hypothesis. Here we use a comparative approach to test one prediction of the hypothesis of aposematism: that coloration will evolve in tandem with toxicity. Recently, we developed a phylogenetic hypothesis of the evolutionary relationships among representative species of poison frogs, using sequences from three regions of mitochondrial DNA. In our analysis, we use that DNA-based phylogeny and comparative analysis of independent contrasts to investigate the correlation between coloration and toxicity in the poison frog family (Dendrobatidae). Information on the toxicity of different species was obtained from the literature. Two different measures of the brightness and extent of coloration were used. (i) Twenty-four human observers were asked to rank different photos of each different species in the analysis in terms of contrast to a leaf-littered background. (ii) Color photos of each species were scanned into a computer and a computer program was used to obtain a measure of the contrast of the colors of each species relative to a leaf-littered background. Comparative analyses of the results were carried out with two different models of character evolution: gradual change, with branch lengths proportional to the amount of genetic change, and punctuational change, with all change being associated with speciation events. Comparative analysis using either method or model indicated a significant correlation between the evolution of toxicity and coloration across this family. These results are consistent with the hypothesis that coloration in this group is aposematic.
Diversity and complexity of HIV-1 drug resistance: A bioinformatics approach to predicting phenotype from genotype
Authors: Beerenwinkel Niko , Schmidt Barbara , Walter Hauke , Kaiser Rolf , Lengauer Thomas , Hoffmann Daniel , Korn Klaus , Selbig Joachim
Published: July 2002

Drug resistance testing has been shown to be beneficial for clinical management of HIV type 1 infected patients. Whereas phenotypic assays directly measure drug resistance, the commonly used genotypic assays provide only indirect evidence of drug resistance, the major challenge being the interpretation of the sequence information. We analyzed the significance of sequence variations in the protease and reverse transcriptase genes for drug resistance and derived models that predict phenotypic resistance from genotypes. For 14 antiretroviral drugs, both genotypic and phenotypic resistance data from 471 clinical isolates were analyzed with a machine learning approach. Information profiles were obtained that quantify the statistical significance of each sequence position for drug resistance. For the different drugs, patterns of varying complexity were observed, including between one and nine sequence positions with substantial information content. Based on these information profiles, decision tree classifiers were generated to identify genotypic patterns characteristic of resistance or susceptibility to the different drugs. We obtained concise and easily interpretable models to predict drug resistance from sequence information. The prediction quality of the models was assessed in leave-one-out experiments in terms of the prediction error. We found prediction errors of 9.6–15.5% for all drugs except for zalcitabine, didanosine, and stavudine, with prediction errors between 25.4% and 32.0%. A prediction service is freely available athttp://cartan.gmd.de/geno2pheno.html.
Identification of the enzymatic mechanism of nitroglycerin bioactivation
Authors: Chen Zhiqiang , Zhang Jian , Stamler Jonathan S.
Published: July 2002

Nitroglycerin (glyceryl trinitrate, GTN), originally manufactured by Alfred Nobel, has been used to treat angina and heart failure for over 130 years. However, the molecular mechanism of GTN biotransformation has remained a mystery and it is not well understood why “tolerance” (i.e., loss of clinical efficacy) manifests over time. Here we purify a nitrate reductase that specifically catalyzes the formation of 1,2-glyceryl dinitrate and nitrite from GTN, leading to production of cGMP and relaxation of vascular smooth muscle both
in vitro
an
d in vivo
, and we identify it as mitochondrial aldehyde dehydrogenase (mtALDH). We also show that mtALDH is inhibited in blood vessels made tolerant by GTN. These results demonstrate that the biotransformation of GTN occurs predominantly in mitochondria through a novel reductase action of mtALDH and suggest that nitrite is an obligate intermediate in generation of NO bioactivity. The data also indicate that attenuated biotransformation of GTN by mtALDH underlies the induction of nitrate tolerance. More generally, our studies provide new insights into subcellular processing of NO metabolites and suggest new approaches to generating NO bioactivity and overcoming nitrate tolerance.
The tight linkage between DNA replication and double-strand break repair in bacteriophage T4
Authors: George James W. , Stohr Bradley A. , Tomso Daniel J. , Kreuzer Kenneth N.
Published: July 2002

Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.
Prediction of cytomegalovirus load and resistance patterns after antiviral chemotherapy
Authors: Emery Vincent C. , Griffiths Paul D.
Published: July 2002

Cytomegalovirus (CMV) replicates rapidly in its human host with a doubling time of ≈ 1 day. Using simple mathematical models we show that the efficacy of the anti-CMV drug ganciclovir (GCV) against wild-type strains is 91.5% [95% confidence interval (CI) 89–94%] when given i.v. (5 mg/kg twice a day) but only 46.5% (95% CI 45–47.5%) when given orally (1 g three times a day) whereas the corresponding figures for a typical GCV-resistant virus are 62% (95% CI 57–66%) and 35% (95% CI 33–37%), respectively. During prolonged periods of GCV therapy we show that the apparent sudden appearance of GCV resistance is explained by the combination of two exponentially increasing populations (wild type and mutant) at doses of GCV that do not completely inhibit CMV replication. Cell culture methods to assess CMV drug resistance
in vivo
will underestimate its prevalence because of the fitness loss of resistant virus in the absence of therapy. The parameters determined from these models then were used to predict the likely viral load and resistance patterns in patients on prolonged therapy with GCV. The modeled and experimental data showed excellent agreement over extended time periods (up to 270 days of therapy) and provide a framework to predict the virologic course of patients at therapeutic initiation.
Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance
Authors: Vancanneyt Guy , Sanz Carlos , Farmaki Theodora , Paneque Manuel , Ortego Félix , Castañera Pedro , Sánchez-Serrano Jose J.
Published: July 2002

Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity
in vitro
and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.
Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-β and Wnt pathways
Authors: Labbé Etienne , Letamendia Ainhoa , Attisano Liliana
Published: July 2002

The transforming growth factor-β (TGFβ) and Wnt/wingless pathways play pivotal roles in tissue specification during development. Activation of Smads, the effectors of TGFβ superfamily signals, results in Smad translocation from the cytoplasm into the nucleus where they act as transcriptional comodulators to regulate target gene expression. Wnt/wingless signals are mediated by the DNA-binding HMG box transcription factors lymphoid enhancer binding factor 1/T cell-specific factor (LEF1/TCF) and their coactivator β-catenin. Herein, we show that Smad3 physically interacts with the HMG box domain of LEF1 and that TGFβ and Wnt pathways synergize to activate transcription of the
Xenopus
homeobox gene twin (
Xtwn
). Disruption of specific Smad and LEF1/TCF DNA-binding sites in the promoter abrogates synergistic activation of the promoter. Consistent with this observation, introduction of Smad sites into a TGFβ-insensitive LEF1/TCF target gene confers cooperative TGFβ and Wnt responsiveness to the promoter. Furthermore, we demonstrate that TGFβ-dependent activation of LEF1/TCF target genes can occur in the absence of β-catenin binding to LEF1/TCF and requires both Smad and LEF1/TCF DNA-binding sites in the Xtwn promoter. Thus, our results show that TGFβ and Wnt signaling pathways can independently or cooperatively regulate LEF1/TCF target genes and suggest a model for how these pathways can synergistically activate target genes.
DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes
Authors: Murray A. E. , Lies D. , Li G. , Nealson K. , Zhou J. , Tiedje J. M.
Published: July 2002

DNA microarrays constructed with full length ORFs from
Shewanella oneidensis
, MR-1, were hybridized with
genomic DNA from nine other
Shewanella
species and
Escherichia coli
K-12. This approach enabled
visualization of relationships between organisms by comparing
individual ORF hybridizations to 164 genes and is further amenable to
high-density high-throughput analyses of complete microbial genomes.
Conserved genes (
arc
A and ATP synthase) were identified
among all species investigated. The
mtr
operon, which is
involved in iron reduction, was poorly conserved among other known
metal-reducing
Shewanella
species. Results were most
informative for closely related organisms with small subunit rRNA
sequence similarities greater than 93% and
gyr
B
sequence similarities greater than 80%. At this level of relatedness,
the similarity between hybridization profiles was strongly correlated
with sequence divergence in the
gyr
B gene. Results
revealed that two strains of
S. oneidensis
(MR-1 and
DLM7) were nearly identical, with only 3% of the ORFs hybridizing
poorly, in contrast to hybridizations with
Shewanella
putrefaciens
, formerly considered to be the same species as
MR-1, in which 63% of the ORFs hybridized poorly (log ratios below
−0.75). Genomic hybridizations showed that genes in operons had
consistent levels of hybridization across an operon in comparison to a
randomly sampled data set, suggesting that similar applications will be
informative for identification of horizontally acquired genes. The
full value of microbial genomic hybridizations lies in providing
the ability to understand and display specific differences between
closely related organisms providing a window into understanding
microheterogeneity, bacterial speciation, and taxonomic relationships.
Shifts in cortical representations predict human discrimination improvement
Authors: Pleger Burkhard , Dinse Hubert R. , Ragert Patrick , Schwenkreis Peter , Malin Jean Pierre , Tegenthoff Martin
Published: July 2002

We report experiments combining assessment of spatial tactile discrimination behavior and measurements of somatosensory-evoked potentials in human subjects before and after short-term plastic changes to demonstrate a causal link between the degree of altered performance and reorganization. Plastic changes were induced by a Hebbian coactivation protocol of simultaneous pairing of tactile stimuli. As a result of coactivation, spatial discrimination thresholds were lowered; however, the amount of discrimination improvement was variable across subjects. Analysis of somatosensory-evoked potentials revealed a significant, but also variable shift in the localization of the N20-dipole of the index finger that was coactivated. The Euclidean distance between the dipole pre- and post-coactivation was significantly larger on the coactivated side (mean 9.13 ± 3.4 mm) than on the control side (mean 4.90 ± 2.7 mm,
P
= 0.008). Changes of polar angles indicated a lateral and inferior shift on the postcentral gyrus of the left hemisphere representing the coactivated index finger. To explore how far the variability of improvement was reflected in the degree of reorganization, we correlated the perceptual changes with the N20-dipole shifts. We found that the changes in discrimination abilities could be predicted from the changes in dipole localization. Little gain in spatial discrimination was associated with small changes in dipole shifts. In contrast, subjects who showed a large cortical reorganization also had lowest thresholds. All changes were highly selective as no transfer to the index finger of the opposite, non-coactivated hand was found. Our results indicate that human spatial discrimination performance is subject to improvement on a short time scale by a Hebbian stimulation protocol without invoking training, attention, or reinforcement. Plastic processes related to the improvement were localized in primary somatosensory cortex and were scaled with the degree of the individual perceptual improvement.
Supramolecular chemistry: Functional structures on the mesoscale
Authors: Nguyen SonBinh T. , Gin Douglas L. , Hupp Joseph T. , Zhang Xi
Published: July 2002

Supramolecular chemistry deals with the chemistry and collective behavior of organized ensembles of molecules. In this so-called mesoscale regime, molecular building blocks are organized into longer-range order and higher-order functional structures via comparatively weak forces. As one of the modern frontiers in chemistry, supramolecular chemistry heralds many promises that range from biocompatible materials and biomimetic catalysts to sensors and nanoscale fabrication of electronic devices.
Demonstration of thein vivointeraction of key cell death regulators by structure-based design of second-site suppressors
Authors: Parrish Jay , Metters Helen , Chen Lin , Xue Ding
Published: July 2002

Demonstratingin vivointeraction of two important biomolecules and the relevance of the interaction to a biological process have been difficult issues in biomedical research. Here, we report the use of a homology modeling approach to establish the significance of protein interactions in governing the activation of programmed cell death inCaenorhabditis elegans.A protein interaction cascade has been postulated to mediate activation of cell death in nematodes, in which the BH3-domain-containing (Bcl-2 homology region 3) protein EGL-1 binds the cell-death inhibitor CED-9 and induces release of the death-activating protein CED-4 from inhibitory CED-4/CED-9 complexes. We show here that an unusual gain-of-function mutation inced-9(substitution of glycine 169 to glutamate) that results in potent inhibition of most nematode cell deaths impairs the binding of EGL-1 to CED-9 and EGL-1-induced release of CED-4 from CED-4/CED-9 complexes. Based on a modeled EGL-1/CED-9 complex structure, we generated second-site compensatory mutations in EGL-1 that partially restore the binding of EGL-1 to CED-9(G169E) and EGL-1-induced release of CED-4 from CED-4/CED-9(G169E) complexes. Importantly, these mutations also significantly suppress the death-protective activity of CED-9(G169E)in vivo. These results establish that direct physical interaction between EGL-1 and CED-9 is essential for the release of CED-4 and the activation of cell death. The structure-based design of second-site suppressors via homology modeling should be widely applicable for probing important molecular interactions that are implicated in fundamental biological processes.
Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA
Authors: Sengupta Jayati , Agrawal Rajendra K. , Frank Joachim
Published: July 2002

S1 is the largest ribosomal protein, present in the small subunit of the bacterial ribosome. It has a pivotal role in stabilizing the mRNA on the ribosome. Thus far, S1 has eluded structural determination. We have identified the S1 protein mass in the cryo-electron microscopic map of theEscherichia coliribosome by comparing the map with a recent x-ray crystallographic structure of the 30S subunit, which lacks S1. According to our finding, S1 is located at the junction of head, platform, and main body of the 30S subunit, thus explaining all existing biochemical and crosslinking data. Protein S1 as identified in our map has a complex, elongated shape with two holes in its central portion. The N-terminal domain, forming one of the extensions, penetrates into the head of the 30S subunit. Evidence for direct interaction of S1 with 11 nucleotides of the mRNA, immediately upstream of the Shine–Dalgarno sequence, explains the protein's role in the recognition of the 5′ region of mRNA.
Structural basis for the activation of human procaspase-7
Authors: Riedl Stefan J. , Fuentes-Prior Pablo , Renatus Martin , Kairies Norman , Krapp Stephan , Huber Robert , Salvesen Guy S. , Bode Wolfram
Published: July 2002

Caspases form a family of proteinases required for the initiation
and execution phases of apoptosis. Distinct
proapoptotic stimuli lead to activation of the initiator
caspases-8 and -9, which in turn activate the common executioner
caspases-3 and -7 by proteolytic cleavage. Whereas crystal structures
of several active caspases have been reported, no three-dimensional
structure of an uncleaved caspase zymogen is available so far. We have
determined the 2.9-Å crystal structure of recombinant human
C285A procaspase-7 and have elucidated the activation
mechanism of caspases. The overall fold of the homodimeric procaspase-7
resembles that of the active tetrameric caspase-7. Each monomer is
organized in two structured subdomains connected by partially flexible
linkers, which asymmetrically occupy and block the central cavity, a
typical feature of active caspases. This blockage is incompatible with
a functional substrate binding site/active site. After proteolytic
cleavage within the flexible linkers, the newly formed chain termini
leave the cavity and fold outward to form stable structures. These
conformational changes are associated with the formation of an intact
active-site cleft. Therefore, this mechanism represents a
formerly unknown type of proteinase zymogen activation.
A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria
Authors: Houman Fariba , Rho Seung Bae , Zhang Jiansu , Shen Xiaoyu , Wang Chien-Chia , Schimmel Paul , Martinis Susan A.
Published: July 2002

Mitochondrial leucyl-tRNA synthetase (LeuRS) in the yeast
Saccharomyces cerevisiae
provides two essential
functions. In addition to aminoacylation, LeuRS functions in RNA
splicing. The details of how it came to act in splicing are not known.
Here we show that
Mycobacterium tuberculosis
and human
mitochondrial LeuRSs can substitute in splicing for the
S.
cerevisiae
mitochondrial LeuRS. Mutations of yeast
mitochondrial LeuRS that had previously been shown to abolish splicing
activity also eliminate splicing by the
M. tuberculosis
enzyme. These results suggest the role of LeuRS in splicing in yeast
mitochondria results from features of the enzyme that are broadly
conserved in evolution. These features are not likely to be designed
for splicing
per se
, but instead have been adopted in
yeast for that purpose.
Integrin α
v
β
3
mediates rotavirus cell entry
Authors: Guerrero Carlos A. , Méndez Ernesto , Zárate Selene , Isa Pavel , López Susana , Arias Carlos F.
Published: July 2002

Rotavirus strains differ in their need for sialic acid (SA) for
initial binding to the cell surface; however, the existence of a
postattachment cell receptor, common to most, if not all, rotavirus
strains, has been proposed. In the present study, antibodies to the
α
v
and β
3
integrin subunits, and the
α
v
β
3
ligand, vitronectin, efficiently
blocked the infectivity of the SA-dependent rhesus rotavirus RRV, its
SA-independent variant nar3, and the neuraminidase-resistant human
rotavirus strain Wa. Vitronectin and anti-β
3
antibodies,
however, did not block the binding of virus to cells, indicating that
rotaviruses interact with α
v
β
3
at a
postbinding step, probably penetration. This interaction was shown to
be independent of the tripeptide motif arginine-glycine-aspartic acid
present in the natural ligands of this integrin. Transfection of CHO
cells with α
v
β
3
genes significantly
increased their permissiveness to all three rotavirus strains, and the
increment of virus infectivity was reverted by incubation of these
cells either with antibodies to β
3
or with vitronectin.
These findings implicate α
v
β
3
integrin as a
cellular receptor common to neuraminidase-sensitive and
neuraminidase-resistant rotaviruses, and support the hypothesis that
this integrin could determine, at least in part, the cellular
susceptibility to rotaviruses.
Plastome-encoded bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco
Authors: Whitney Spencer M. , Andrews T. John
Published: July 2002

The efficiency with which crop plants use their resources of light,
water, and fertilizer nitrogen could be enhanced by replacing
their CO
2
-fixing enzyme,
d
-ribulose-1,5-bisphosphate carboxylase-oxygenase
(RubisCO), with more efficient forms, such as those found in some
algae, for example. This important challenge has been frustrated by
failure of all previous attempts to substitute a fully functional,
foreign RubisCO (efficient or inefficient) into higher plants. This
failure could be caused by incompatibility between the plastid-encoded
large subunits and the nucleus-encoded small subunits or by inability
of the foreign RubisCO subunits to fold or assemble efficiently in the
plastid. Mismatch between the regulatory requirements of the foreign
RubisCO and conditions in the chloroplast also might render the
substituted enzyme inactive but, previously, it has not been possible
to test this. To answer the general question of whether a foreign
RubisCO can support photosynthesis in a plant, we used plastid
transformation to replace RubisCO in tobacco with the simple
homodimeric form of the enzyme from the α-proteobacterium,
Rhodospirillum rubrum
, which has no small subunits and
no special assembly requirements. The transplastomic plants so obtained
are fully autotrophic and reproductive but require CO
2
supplementation, consistent with the kinetic properties of the
bacterial RubisCO. This establishes that the activity of a RubisCO from
a very different phylogeny can be integrated into chloroplast
photosynthetic metabolism without prohibitive problems.
Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation.
Authors: Leonard B A , Podbielski A , Hedberg P J , Dunny G M
Published: July 2002

Conjugative transfer of the plasmid pCF10 by Enterococcus faecalis donor cells occurs in response to a peptide sex pheromone, cCF10, secreted by recipients. The plasmid-encoded cCF10 binding protein, PrgZ, is similar in sequence to binding proteins (OppAs) encoded by oligopeptide permease (opp) operons. Mutation of prgZ decreased the sensitivity of donor cells to pheromone, whereas inactivation of the chromosomal E. faecalis opp operon abolished response at physiological concentrations of pheromone. Affinity chromatography experiments demonstrated the interaction of the pheromone with several putative intracellular regulatory molecules, including an RNA molecule required for positive regulation of conjugation functions. These data suggest that processing of the pheromone signal involves recruitment of a chromosomal Opp system by PrgZ and that signaling occurs by direct interaction of internalized pheromone with intracellular effectors.
Activated Ras signals differentiation and expansion of CD4+8+ thymocytes.
Authors: Swat W , Shinkai Y , Cheng H L , Davidson L , Alt F W
Published: July 2002

We describe a novel approach to assay the ability of particular gene products to signal transitions in lymphocyte differentiation in vivo. The method involves transfection of test expression constructs into RAG-1-deficient embryonic stem cells, which are subsequently assayed by the RAG-2-deficient blastocyst complementation approach. We have used this method to demonstrate that expression of activated Ras in CD4-8- (double negative, DN) prothymocytes in vivo induces their differentiation into small CD4+8+ (double positive, DP) cortical thymocytes with accompanying expansion to normal thymocyte numbers. However, activated Ras expression in DP cells does not cause proliferation or maturation to CD4+8- or CD4-8+ (single positive) thymocytes. Therefore, signaling through Ras is sufficient for promoting differentiation of DN to DP cells, but further differentiation requires the activity of additional signaling pathways.