bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleoprotein-based nanoscale assembly
Authors: Smith Steven S. , Niu Luming , Baker David J. , Wendel John A. , Kane Susan E. , Joy Darrin S.
Published: July 2002

A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-fluorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M·
Hha
I and M·
Msp
I. When 5-fluorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M·
Hha
I or FCGG for M·
Msp
I), we show that the first recognition sequence becomes an address for M·
Hha
I, while the second sequence becomes an address for M·
Msp
I. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M·
Hha
I retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein–DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.
Regulation of intestinal uroguanylin/guanylin receptor-mediated responses by mucosal acidity
Authors: Hamra F. Kent , Eber Sammy L. , Chin David T. , Currie Mark G. , Forte Leonard R.
Published: July 2002

Guanylin and uroguanylin are intestinal peptides that stimulate chloride secretion by activating a common set of receptor–guanylate cyclase signaling molecules located on the mucosal surface of enterocytes. High mucosal acidity, similar to the pH occurring within the fluid microclimate domain at the mucosal surface of the intestine, markedly enhances the cGMP accumulation responses of T84 human intestinal cells to uroguanylin. In contrast, a mucosal acidity of pH 5.0 renders guanylin essentially inactive. T84 cells were used as a model epithelium to further explore the concept that mucosal acidity imposes agonist selectivity for activation of the intestinal receptors for uroguanylin and guanylin, thus providing a rationale for the evolution of these related peptides. At an acidic mucosal pH of 5.0, uroguanylin is 100-fold more potent than guanylin, but at an alkaline pH of 8.0 guanylin is more potent than uroguanylin in stimulating intracellular cGMP accumulation and transepithelial chloride secretion. The relative affinities of uroguanylin and guanylin for binding to receptors on the mucosal surface of T84 cells is influenced dramatically by mucosal acidity, which explains the strong pH dependency of the cGMP and chloride secretion responses to these peptides. The guanylin-binding affinities for peptide–receptor interaction were reduced by 100-fold at pH 5 versus pH 8, whereas the affinities of uroguanylin for these receptors were increased 10-fold by acidic pH conditions. Deletion of the N-terminal acidic amino acids in uroguanylin demonstrated that these residues are responsible for the increase in binding affinities that are observed for uroguanylin at acidic pH. We conclude that guanylin and uroguanylin evolved distinctly different structures, which enables both peptides to regulate, in a pH-dependent fashion, the activity of receptors that control intestinal salt and water transport via cGMP.
A proliferation switch for genetically modified cells
Authors: Blau C. Anthony , Peterson Kenneth R. , Drachman Jonathan G. , Spencer David M.
Published: July 2002

Receptor dimerization is the key signaling event for many cytokines, including erythropoietin. A system has been recently developed that permits intracellular protein dimerization to be reversibly activated in response to a lipid-soluble dimeric form of the drug FK506, called FK1012. FK1012 is used as a pharmacological mediator of dimerization to bring together FK506 binding domains, taken from the endogenous protein FKBP12. In experiments reported herein, FK1012-induced dimerization of a fusion protein containing the intracellular portion of the erythropoietin receptor allowed cells normally dependent on interleukin 3 to proliferate in its absence. FK506 competitively reversed the proliferative effect of FK1012 but had no influence on the proliferative effect of interleukin 3. Signaling pathways activated by FK1012 mimicked those activated by erythropoietin, because both JAK2 and STAT5 were phosphorylated in response to FK1012. This approach may provide a means to specifically and reversibly stimulate the proliferation of genetically modified cell populations
in vitro
or
in vivo
.
Identification of sulfated oligosialic acid units in theO-linked glycan of the sea urchin egg receptor for sperm
Authors: Kitazume-Kawaguchi Shinobu , Inoue Sadako , Inoue Yasuo , Lennarz William J.
Published: July 2002

TheStrongylocentrotus purpuratussea urchin egg receptor for sperm is a cell surface glycoprotein with a molecular mass of 350 kDa. Recent studies indicate that the sulfatedO-linked glycans isolated from the receptor bind to acrosome-reacted sperm. The purified receptor was analyzed with respect to amino acid and carbohydrate content and shown to be composed of 70% carbohydrate by weight. Compositional analysis indicated that bothN- andO-linked oligosaccharide chains were present. After peptide:N-glycanase treatment of the receptor to remove most of theN-linked glycan chains, the majority of the sialic acid residues remained associated with the receptor and were shown by several types of experiments to be composed of sulfated oligosialic acid units attached to theO-linked glycan chains of the receptor. Chemical and physical studies on oligosialic chains discovered earlier in the Pronase-generated glycopeptide fraction isolated from the egg cell surface complex of another species of sea urchin,Hemicentrotus pulcherrimus, established that these molecules had the structure: (SO4−)-9Neu5Gcα2(→5-OglycolylNeu5Gcα2→)n. Based on comparative and analytical studies, it was concluded that this sulfated oligosaccharide is a component of a GalNAc-containing chain that isO-linked to the polypeptide chain of the sea urchin egg receptor for sperm. Using a competitive inhibition of fertilization bioassay it was shown that the sulfated oligosialic acid chains derived from theS. purpuratusegg cell surface complex inhibited fertilization; the nonsulfated form of this oligosialic chain had little inhibitory activity.
Measuring cosmological parameters
Authors: Freedman Wendy L.
Published: July 2002

In this review, the status of measurements of the matter density
(Ω
m
), the vacuum energy density or cosmological constant
(Ω
Λ
), the Hubble constant
(
H
0
), and the ages of the oldest measured
objects (
t
0
) are summarized. Three
independent types of methods for measuring the Hubble constant are
considered: the measurement of time delays in multiply imaged quasars,
the Sunyaev–Zel’dovich effect in clusters, and Cepheid-based
extragalactic distances. Many recent independent dynamical measurements
are yielding a low value for the matter density (Ω
m
≈
0.2–0.3). A wide range of Hubble constant measurements appear to be
converging in the range of 60–80 km/sec per megaparsec. Areas
where future improvements are likely to be made soon are
highlighted—in particular, measurements of anisotropies in the
cosmic microwave background. Particular attention is paid to sources of
systematic error and the assumptions that underlie many of the
measurement methods.
Morphine disrupts long-range synchrony of gamma oscillations in hippocampal slices
Authors: Whittington M. A. , Traub R. D. , Faulkner H. J. , Jefferys J. G. R. , Chettiar K.
Published: July 2002

Oscillations in neuronal population activity within the gamma frequency band (>25 Hz) have been correlated with cognition: Gamma oscillations could bind together features of a sensory stimulus by generating synchrony between discrete cortical areas [Eckhorn, R., Bauer, R., Jordan, W., Brosch, M., Kruse, W., Munk, M. & Reitboeck, H. J. (1989)
Biol. Cybern.
60, 121–130; Singer, W. & Gray, C. M. (1995)
Annu. Rev. Neurosci.
18, 555–556]. Herein we demonstrate that morphine and β-endorphin disrupt this long-range synchrony of gamma oscillations while leaving the synchrony of local oscillations relatively intact. The effect is caused by a decrease in type A γ-aminobutyric acid receptor-mediated inhibition of both excitatory pyramidal cells and inhibitory interneurons. The effects of morphine on gamma oscillations were blocked by μ-opioid receptor antagonists but not by antagonists of δ or κ receptors. Morphine also produced burst firing in interneurons, because synaptic excitation from pyramidal cells was no longer balanced by synchronous inhibitory postsynaptic potentials. The loss of synchrony of gamma oscillations induced by morphine may constitute one mechanism involved in producing the cognitive deficits that this drug causes clinically.
The interspecific mass–density relationship and plant geometry
Authors: Franco Miguel , Kelly Colleen K.
Published: July 2002

We present ana prioritheoretical framework for the interspecific allometric relationship between stand mass and plant population density. Our model predicts a slope of −\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\frac{1}{3}\end{equation*}\end{document}between the logarithm of stand mass and the logarithm of stand density, thus conflicting with a previously assumed slope of −½. Our model rests on a heuristic separation of resource-limited living mass and structural mass in the plant body. We point out that because of similar resource requirements among plants of different sizes, a nonzero plant mass–density slope is primarily defined by structural mass. Specifically, the slope is a result of (i) the physical size-dependent relationship between stem width and height, (ii) foliage-dependent demands of conductance, and (iii) the cumulative nature of structural mass. The data support our model, both when the potential sampling bias of taxonomic relatedness is accounted for and when it is not. Independent contrasts analyses show that observed relationships among variables are not significantly different from the assumptions made to build the model or from itsa prioripredictions. We note that the dependence of the plant mass–density slope on the functions of structural mass provides a cause for the difference from the zero slope found in the animal population mass–density relationship; for the most part, animals do not have a comparable cumulative tissue type.
A member of a family of sulfate-activating enzymes causes murine brachymorphism
Authors: Kurima Kiyoto , Warman Matthew L. , Krishnan Srinivasan , Domowicz Miriam , Krueger Richard C. , Deyrup Andrea , Schwartz Nancy B.
Published: July 2002

Sulfation is critical to the function of a wide variety of biomolecules. This common modification requires the enzymatic synthesis of an activated sulfate donor, phosphoadenosine-phosphosulfate (PAPS). In higher organisms PAPS synthesis is catalyzed by a bifunctional sulfurylase kinase (SK) polypeptide having both ATP-sulfurylase and adenosine-phosphosulfate kinase activities. We report the identification of a gene family encoding murine SK proteins with these two activities. A family member,
SK2
, colocalizes with the locus for the autosomal recessive murine phenotype brachymorphism. Brachymorphic mice have normal lifespans, but abnormal hepatic detoxification, bleeding times, and postnatal growth, the latter being attributed to undersulfation of cartilage proteoglycan. A missense mutation in the
SK2
coding sequence of
bm
mice that alters a highly conserved amino acid residue destroys adenosine-phosphosulfate kinase activity and therefore the ability of SK2 to synthesize PAPS. We conclude that a family of
SK
genes are responsible for sulfate activation in mammals, that a mutation in
SK2
causes murine brachymorphism, and that members of this gene family have nonredundant, tissue-specific roles.
Evidence for gene silencing by endogenous DNA methylation
Authors: Holliday Robin , Ho Thu
Published: July 2002

Transformed cells can spontaneously silence genes by
de novo
methylation, and it is generally assumed that this is due to DNA methyltransferase activity. We have tested the alternative hypothesis that gene silencing could be due to the uptake of 5-methyl-dCMP into DNA, via the di- and triphosphonucleotides. 5-Methyl-dCMP would be present in cells from the ongoing repair of DNA. We have isolated a strain of Chinese hamster ovary (CHO) cells, designated HAM
−
, which spontaneously silences two tested genes at a very high frequency. We have shown that this strain incorporates 5-[
3
H]methyldeoxycytidine into 5-methylcytosine and thymine in DNA. It also has low 5-methyl-dCMP deaminase activity. Another HAM
+
strain has high deaminase activity and a very low frequency of gene silencing. The starting strain, CHO K1, has a phenotype intermediate between HAM
−
and HAM
+
.
Three new structures of the core domain of HIV-1 integrase: An active site that binds magnesium
Authors: Goldgur Yehuda , Dyda Fred , Hickman Alison B. , Jenkins Timothy M. , Craigie Robert , Davies David R.
Published: July 2002

HIV-1 integrase is an essential enzyme in the life cycle of the virus, responsible for catalyzing the insertion of the viral genome into the host cell chromosome; it provides an attractive target for antiviral drug design. The previously reported crystal structure of the HIV-1 integrase core domain revealed that this domain belongs to the superfamily of polynucleotidyltransferases. However, the position of the conserved catalytic carboxylic acids differed from those observed in other enzymes of the class, and attempts to crystallize in the presence of the cofactor, Mg
2+
, were unsuccessful. We report here three additional crystal structures of the core domain of HIV-1 integrase mutants, crystallized in the presence and absence of cacodylate, as well as complexed with Mg
2+
. These three crystal forms, containing between them seven independent core domain structures, demonstrate the unambiguous extension of the previously disordered helix α4 toward the amino terminus from residue M154 and show that the catalytic E152 points in the general direction of the two catalytic aspartates, D64 and D116. In the vicinity of the active site, the structure of the protein in the absence of cacodylate exhibits significant deviations from the previously reported structures. These differences can be attributed to the modification of C65 and C130 by cacodylate, which was an essential component of the original crystallization mixture. We also demonstrate that in the absence of cacodylate this protein will bind to Mg
2+
, and could provide a satisfactory platform for binding of inhibitors.
Self-identification of protein-coding regions in microbial genomes
Authors: Audic Stéphane , Claverie Jean-Michel
Published: July 2002

A new method for predicting protein-coding regions in microbial genomic DNA sequences is presented. It uses an
ab initio
iterative Markov modeling procedure to automatically perform the partition of genomic sequences into three subsets shown to correspond to coding, coding on the opposite strand, and noncoding segments. In contrast to current methods, such as
genemark
[Borodovsky, M. & McIninch, J. D. (1993)
Comput. Chem.
17, 123–133], no training set or prior knowledge of the statistical properties of the studied genome are required. This new method tolerates error rates of 1–2% and can process unassembled sequences. It is thus ideal for the analysis of genome survey and/or fragmented sequence data from uncharacterized microorganisms. The method was validated on 10 complete bacterial genomes (from four major phylogenetic lineages). The results show that protein-coding regions can be identified with an accuracy of up to 90% with a totally automated and objective procedure.
Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose
Authors: Durner Jörg , Wendehenne David , Klessig Daniel F.
Published: July 2002

Reactive oxygen species are believed to perform multiple roles during plant defense responses to microbial attack, acting in the initial defense and possibly as cellular signaling molecules. In animals, nitric oxide (NO) is an important redox-active signaling molecule. Here we show that infection of resistant, but not susceptible, tobacco with tobacco mosaic virus resulted in enhanced NO synthase (NOS) activity. Furthermore, administration of NO donors or recombinant mammalian NOS to tobacco plants or tobacco suspension cells triggered expression of the defense-related genes encoding pathogenesis-related 1 protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cyclic GMP (cGMP) and cyclic ADP-ribose, two molecules that can serve as second messengers for NO signaling in mammals. Consistent with cGMP acting as a second messenger in tobacco, NO treatment induced dramatic and transient increases in endogenous cGMP levels. Furthermore, NO-induced activation of
PAL
was blocked by 6-anilino-5,8-quinolinedione and 1
H
-(1,2,4)-oxadiazole[4,3-
a
]quinoxalin-1-one, two inhibitors of guanylate cyclase. Although 6-anilino-5,8-quinolinedione fully blocked
PAL
activation, inhibition by 1
H
-(1,2,4)-oxadiazole[4,3-
a
]quinoxalin-1-one was not entirely complete, suggesting the existence of cGMP-independent, as well as cGMP-dependent, NO signaling. We conclude that several critical players of animal NO signaling are also operative in plants.
Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase
Authors: Hasson Miriam Sarah , Schlichting Ilme , Moulai Javad , Taylor Kirk , Barrett William , Kenyon George L. , Babbitt Patricia C. , Gerlt John A. , Petsko Gregory A. , Ringe Dagmar
Published: July 2002

Muconate lactonizing enzyme (MLE), a component of the β-ketoadipate pathway of
Pseudomonas putida
, is a member of a family of related enzymes (the “enolase superfamily”) that catalyze the abstraction of the α-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-Å resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn
2+
-bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (
i
) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (
ii
) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.
In vivomagnetic resonance vascular imaging using laser-polarized3He microbubbles
Authors: Chawla Mark S. , Chen X. Josette , Möller Harald E. , Cofer Gary P. , Wheeler C. Ted , Hedlund Laurence W. , Johnson G. Allan
Published: July 2002

Laser-polarized gases (3He and129Xe) are currently being used in magnetic resonance imaging as strong signal sources that can be safely introduced into the lung. Recently, researchers have been investigating other tissues using129Xe. These studies use xenon dissolved in a carrier such as lipid vesicles or blood. Since helium is much less soluble than xenon in these materials,3He has been used exclusively for imaging air spaces. However, considering that the signal of3He is more than 10 times greater than that of129Xe for presently attainable polarization levels, this work has focused on generating a method to introduce3He into the vascular system. We addressed the low solubility issue by producing suspensions of3He microbubbles. Here, we provide the first vascular images obtained with laser-polarized3He. The potential increase in signal and absence of background should allow this technique to produce high-resolution angiographic images. In addition, quantitative measurements of blood flow velocity and tissue perfusion will be feasible.
An extension of the steepest descent method for Riemann-Hilbert problems: The small dispersion limit of the Korteweg-de Vries (KdV) equation
Authors: Deift P. , Venakides S. , Zhou X.
Published: July 2002

This paper extends the steepest descent method for Riemann-Hilbert
problems introduced by Deift and Zhou in a critical new way. We
present, in particular, an algorithm, to obtain the support of the
Riemann-Hilbert problem for leading asymptotics. Applying this extended
method to small dispersion KdV (Korteweg-de Vries) equation, we
(
i
) recover the variational formulation of P. D.
Lax and C. D. Levermore [(1979)
Proc. Natl. Acad. Sci. USA
76, 3602–3606] for the weak limit of the solution,
(
ii
) derive, without using an ansatz, the hyperelliptic
asymptotic solution of S. Venakides that describes the oscillations;
and (
iii
) are now able to compute the phase shifts,
integrating the modulation equations exactly. The procedure of this
paper is a version of fully nonlinear geometrical optics for integrable
systems. With some additional analysis the theory can provide rigorous
error estimates between the solution and its computed asymptotic
expression.
Synthesis of medium-chain-length polyhydroxyalkanoates inArabidopsis thalianausing intermediates of peroxisomal fatty acid β-oxidation
Authors: Mittendorf Volker , Robertson Elizabeth J. , Leech Rachel M. , Krüger Niels , Steinbüchel Alexander , Poirier Yves
Published: July 2002

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the β-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants,Arabidopsis thalianawas transformed with the PhaC1 synthase fromPseudomonas aeruginosamodified for peroxisome targeting by addition of the carboxyl 34 amino acids from theBrassica napusisocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the β-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.
Targeting cyclin-dependent kinases in
Drosophila
with peptide aptamers
Authors: Kolonin Mikhail G. , Finley Russell L.
Published: July 2002

Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as
in vivo
inhibitors. Here we explore the ability to use peptide aptamers as
in vivo
inhibitors by expressing aptamers directed against cell cycle regulators in
Drosophila
. We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in
Drosophila
. Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition. Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target
in vivo
and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function. Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in
Drosophila
. Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins
in vivo
.
Prolonged activation of the
N
-methyl-
d
-aspartate receptor–Ca
2+
transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture
Authors: DeLorenzo Robert J. , Pal Shubhro , Sombati Sompong
Published: July 2002

The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca
2+
-dependent. The concentration of intracellular free Ca
2+
([Ca
2+
]
i
) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg
2+
treatment by confocal laser-scanning fluorescent microscopy to directly correlate changes in [Ca
2+
]
i
with alterations in membrane excitability measured by intracellular recording using whole-cell current–clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg
2+
-induced spike discharges, was prevented in low-Ca
2+
solutions and was dependent on activation of the
N
-methyl-
d
-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA–Ca
2+
transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.
Anatomy of word and sentence meaning
Authors: Posner Michael I. , Pavese Antonella
Published: July 2002

Reading and listening involve complex psychological processes that
recruit many brain areas. The anatomy of processing English words has
been studied by a variety of imaging methods. Although there is
widespread agreement on the general anatomical areas involved in
comprehending words, there are still disputes about the computations
that go on in these areas. Examination of the time relations
(circuitry) among these anatomical areas can aid in understanding their
computations. In this paper, we concentrate on tasks that involve
obtaining the meaning of a word in isolation or in relation to a
sentence. Our current data support a finding in the literature that
frontal semantic areas are active well before posterior areas. We use
the subject’s attention to amplify relevant brain areas involved
either in semantic classification or in judging the relation of the
word to a sentence to test the hypothesis that frontal areas are
concerned with lexical semantics and posterior areas are more involved
in comprehension of propositions that involve several words.
Grass genomes
Authors: Bennetzen Jeffrey L. , SanMiguel Phillip , Chen Mingsheng , Tikhonov Alexander , Francki Michael , Avramova Zoya
Published: July 2002

For the most part, studies of grass genome structure have been
limited to the generation of whole-genome genetic maps or the fine
structure and sequence analysis of single genes or gene clusters. We
have investigated large contiguous segments of the genomes of maize,
sorghum, and rice, primarily focusing on intergenic spaces. Our data
indicate that much (>50%) of the maize genome is composed of
interspersed repetitive DNAs, primarily nested retrotransposons that
insert between genes. These retroelements are less abundant in smaller
genome plants, including rice and sorghum. Although 5- to 200-kb blocks
of methylated, presumably heterochromatic, retrotransposons flank most
maize genes, rice and sorghum genes are often adjacent. Similar genes
are commonly found in the same relative chromosomal locations and
orientations in each of these three species, although there are
numerous exceptions to this collinearity (i.e., rearrangements) that
can be detected at the levels of both the recombinational map and
cloned DNA. Evolutionarily conserved sequences are largely confined to
genes and their regulatory elements. Our results indicate that a
knowledge of grass genome structure will be a useful tool for gene
discovery and isolation, but the general rules and biological
significance of grass genome organization remain to be determined.
Moreover, the nature and frequency of exceptions to the general
patterns of grass genome structure and collinearity are still largely
unknown and will require extensive further investigation.
Directed changes in the number of double-stranded RNA genomic segments in bacteriophage Φ6
Authors: Onodera Shiroh , Qiao Xueying , Qiao Jian , Mindich Leonard
Published: July 2002

Bacteriophage Φ6 has a genome of three segments of double-stranded RNA. The segments are designated S, M, and L. Each segment has a unique packaging site,
pac,
near the 5′ end of the plus strand. The plus strands of the segments are normally packaged in the order S, M, L. Chimeras of segment M and S in which segment M is at the 5′ end of the plus strand can be stably incorporated into the virion; however, an independent segment S must be included along with normal segment L, even if it contains no active genes. A chimera of segment M and S in which segment S is at the 5′ end of the plus strand can be stably incorporated into the virion along with normal segment L to form a two-segment genome. A chimera of segments S, M, and L in which the packaging sequence is that of S can also form a stable nonsegmented genome. These findings are consistent with a model that we have proposed for the packaging of the Φ6 genome.
Numerical simulation of incompressible viscous flow in deforming domains
Authors: Colella Phillip , Trebotich David P.
Published: July 2002

We present a second-order accurate finite difference method for numerical solution of the incompressible Navier-Stokes equations in deforming domains. Our approach is a generalization of the Bell-Colella-Glaz predictor–corrector method for incompressible flow. In order to treat the time-dependence and inhomogeneities in the incompressibility constraint introduced by presence of deforming boundaries, we introduce a nontrivial splitting of the velocity field into vortical and potential components to eliminate the inhomogeneous terms in the constraint and a generalization of the Bell-Colella-Glaz algorithm to treat time-dependent constraints. The method is second-order accurate in space and time, has a time step constraint determined by the advective Colella-Friedrichs-Lewy condition, and requires the solution of well behaved linear systems amenable to the use of fast iterative methods. We demonstrate the method on the specific example of viscous incompressible flow in an axisymmetric deforming tube.
Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove
Authors: Takebayashi Yuji , Pourquier Philippe , Yoshida Akira , Kohlhagen Glenda , Pommier Yves
Published: July 2002

Ecteinascidin 743 (Et743, National Service Center 648766) is a potent antitumor agent from the Caribbean tunicate
Ecteinascidia turbinata
. Although Et743 is presently in clinical trials for human cancers, the mechanisms of antitumor activity of Et743 have not been elucidated. Et743 can alkylate selectively guanine N2 from the DNA minor groove, and this alkylation is reversed by DNA denaturation. Thus, Et743 differs from other DNA alkylating agents presently in the clinic (by both its biochemical activities and its profile of antitumor activity in preclinical models). In this study, we investigated cellular proteins that can bind to DNA alkylated by Et743. By using an oligonucleotide containing high-affinity Et743 binding sites and nuclear extracts from human leukemia CEM cells, we purified a 100-kDa protein as a cellular target of Et743 and identified it as topoisomerase I (top1). Purified top1 was then tested and found to produce cleavage complexes in the presence of Et743, whereas topoisomerase II had no effect. DNA alkylation was essential for the formation of top1-mediated cleavage complexes by Et743, and the distribution of the drug-induced top1 sites was different for Et743 and camptothecin. top1–DNA complexes were also detected in Et743-treated CEM cells by using cesium chloride gradient centrifugation followed by top1 immunoblotting. These data indicate that DNA minor groove alkylation by Et743 induces top1-mediated protein-linked DNA breaks and that top1 is a target for Et743
in vitro
and
in vivo
.
Receptor recruitment: A mechanism for interactions between G protein-coupled receptors
Authors: Holtbäck Ulla , Brismar Hjalmar , DiBona Gerald F. , Fu Michael , Greengard Paul , Aperia Anita
Published: July 2002

There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the cell to the plasma membrane. The results indicate that recruitment of G protein-coupled receptors may be a ubiquitous mechanism for receptor sensitization and may play a role in the modulation of signal transduction comparable to that of the well established phenomenon of receptor endocytosis and desensitization.
VirulentSalmonella typhimuriumhas two periplasmic Cu, Zn-superoxide dismutases
Authors: Fang Ferric C. , DeGroote Mary Ann , Foster John W. , Bäumler Andreas J. , Ochsner Urs , Testerman Traci , Bearson Shawn , Giárd Jean-Christophe , Xu Yisheng , Campbell Gail , Laessig Tracey
Published: July 2002

Periplasmic Cu, Zn-cofactored superoxide dismutase (SodC) protects Gram-negative bacteria from exogenous oxidative damage. The virulentSalmonella typhimuriumstrain ATCC 14028s has been found to contain two discrete periplasmic Cu, Zn-SOD enzymes that are only 57% identical at the amino acid level. SodCI is carried by a cryptic bacteriophage, and SodCII is closely related to the Cu, Zn-superoxide dismutase ofEscherichia coli. AllSalmonellaserotypes appear to carry thesodCIIlocus, but the phage-associatedsodCIgene is found only in certain strains belonging to the most highly pathogenic serotypes. Expression of eithersodClocus appears to be enhanced during stationary phase, but onlysodCIIis regulated by the alternative sigma factor σs(RpoS). Mutants lacking bothsodCgenes are less lethal for mice than mutants possessing eithersodClocus alone, indicating that both Cu, Zn-SOD enzymes contribute toSalmonellapathogenicity. The evolutionary acquisition of an additionalsodCgene has contributed to the enhanced virulence of selectedSalmonellastrains.
The lack of consensus for I-Ag7-peptide binding motifs: Is there a requirement for anchor amino acid side chains?
Authors: Carrasco-Marin Eugenio , Kanagawa Osami , Unanue Emil R.
Published: July 2002

We discuss here the problems in identifying sequence motifs of peptides that bind to I-Ag7, the class II histocompatibility molecule of NOD diabetic mice. We present studies that indicate a minor contribution of amino acid side chains for binding. A peptide from the Eα chain binds to I-Ag7molecules and is recognized by CD4 T cells. By producing single-residue mutations we identified four residues that were considered to contact the T cell receptor. No residue was found to be essential for binding to I-Ag7: a peptide that contained the T cell contact residues, on a backbone of alanines, bound to I-Ag7and stimulated the T cells. We conclude that peptides can bind to I-Ag7without the requirement for residues with prominent side chains to anchor them.
High expression of a specific T-cell receptor γ transcript in epithelial cells of the prostate
Authors: Essand Magnus , Vasmatzis George , Brinkmann Ulrich , Duray Paul , Lee Byungkook , Pastan Ira
Published: July 2002

We have identified expression of T-cell receptor γ chain (TCRγ) mRNA in human prostate and have shown that it originates from epithelial cells of the prostate and not from infiltrating T-lymphocytes. In contrast, the T-cell receptor δ chain (TCRδ) gene is silent in human prostate. The major TCRγ transcript in prostate has a different size than the transcript expressed in thymus, spleen, and blood leukocytes. It is expressed in normal prostate epithelium, adenocarcinoma of the prostate, and the prostatic adenocarcinoma cell line LNCaP. The RNA originates from an unrearranged TCRγ locus, and it is initiated within the intronic sequence directly upstream of the Jγ1.2 gene segment. The prostate-specific TCRγ transcript consists of the Jγ1.2 and Cγ1 gene segments, and it has an untranslated sequence including a polyadenylation signal and poly(A) sequence at the 3′end. The finding that prostate epithelial cells express a high level of a transcript from a gene that was thought to by exclusively expressed by T-lymphocytes is highly unexpected.
Pearling in cells: A clue to understanding cell shape
Authors: Bar-Ziv Roy , Tlusty Tsvi , Moses Elisha , Safran Samuel A. , Bershadsky Alexander
Published: July 2002

Gradual disruption of the actin cytoskeleton induces a series of
structural shape changes in cells leading to a transformation of
cylindrical cell extensions into a periodic chain of “pearls.”
Quantitative measurements of the pearling instability give a
square-root behavior for the wavelength as a function of drug
concentration. We present a theory that explains these observations in
terms of the interplay between rigidity of the submembranous actin
shell and tension that is induced by boundary conditions set by
adhesion points. The theory allows estimation of the rigidity and
thickness of this supporting shell. The same theoretical considerations
explain the shape of nonadherent edges in the general case of untreated
cells.
Competition for antigenic sites during T cell proliferation: A mathematical interpretation ofin vitrodata
Authors: Borghans José A. M. , Taams Leonie S. , Wauben Marca H. M. , de Boer Rob J.
Published: July 2002

By fitting different mathematical T cell proliferation functions toin vitroT cell proliferation data, we studied T cell competition for stimulatory signals. In our lymphocyte proliferation assays both the antigen (Ag) availability and the concentration of T cells were varied. We show that proliferation functions involving T cell competition describe the data significantly better than classical proliferation functions without competition, thus providing direct evidence for T cell competitionin vitro. Our mathematical approach allowed us to study the nature of T cell competition by comparing different proliferation functions involving (i) direct inhibitory T–T interactions, (ii) Ag-specific resource competition, or (iii) resource competition for nonspecific factors such as growth factors, and access to the surface of Ag-presenting cells (APCs). We show that resource competition is an essential ingredient of T cell proliferation. To discriminate between Ag-specific and nonspecific resource competition, the Ag availability was varied in two manners. In a first approach we varied the concentration of APCs, displaying equal ligand densities; in a second approach we varied the Ag density on the surface of the APCs, while keeping the APC concentration constant. We found that both resource competition functions described the data equally well when the Ag availability was increased by adding APCs. When the APC concentration was kept constant, the nonspecific resource competition function yielded the best description of the data. Our interpretation is that T cells were competing for “antigenic sites” on the APCs.
New estimates of the rates and effects of mildly deleterious mutation in
Drosophila melanogaster
Authors: Fry James D. , Keightley Peter D. , Heinsohn Stefanie L. , Nuzhdin Sergey V.
Published: July 2002

The genomic rate and distribution of effects of deleterious mutations are important parameters in evolutionary theory. The most detailed information comes from the work of Mukai and Ohnishi, who allowed mutations to accumulate on
Drosophila melanogaster
second chromosomes, shielded from selection and recombination by being maintained heterozygous in males. Averaged over studies, the estimated rate of nonlethal viability mutations per second chromosome per generation under an equal-effects model,
U
BM
, was 0.12, suggesting a high genomic mutation rate. We have performed a mutation-accumulation experiment similar to those of Mukai and Ohnishi, except that three large homozygous control populations were maintained. Egg-to-adult viability of 72 nonlethal mutation-accumulation (MA) lines and the controls was assayed after 27–33 generations of mutation accumulation. The rate of decline in mean viability was significantly lower than observed by Mukai, and the rate of increase in among-line variance was significantly higher. Our
U
BM
estimate of 0.02 is much lower than the previous estimates. Our results suggest that the rate of mutations that detectably reduce viability may not be much greater than the lethal mutation rate (0.01 in these lines), but the results also are consistent with models that include many mutations with very small effects.
Verification of phylogenetic predictions
in vivo
and the importance of the tetraloop motif in a catalytic RNA
Authors: Krummel Daniel A. Pomeranz , Altman Sidney
Published: July 2002

M1 RNA, the catalytic subunit of
Escherichia coli
RNase P, forms a secondary structure that includes five sequence variants of the tetraloop motif. Site-directed mutagenesis of the five tetraloops of M1 RNA, and subsequent steady-state kinetic analysis
in vitro
, with different substrates in the presence and absence of the protein cofactor, reveal that (
i
) certain mutants exhibit defects that vary in a substrate-dependent manner, and that (
ii
) the protein cofactor can correct the mutant phenotypes
in vitro
, a phenomenon that is also substrate dependent. Thermal denaturation curves of tetraloop mutants that exhibit kinetic defects differ from those of wild-type M1 RNA. Although the data collected
in vitro
underscore the importance of the tetraloop motif to M1 RNA function and structure, three of the five tetraloops we examined
in vivo
are essential for the function of
E. coli
RNase P. The kinetic data
in vitro
are not in total agreement with previous phylogenetic predictions but the data
in vivo
are, as only mutants in those tetraloops proposed to be involved in tertiary interactions fail to complement
in vivo
. Therefore, the tetraloop motif is critical for the stabilization of the structure of M1 RNA and essential to RNase P function in the cell.
Human ATP-binding cassette transporter 1 (ABC1): Genomic organization and identification of the genetic defect in the original Tangier disease kindred
Authors: Remaley Alan T. , Rust Stephan , Rosier Marie , Knapper Cathy , Naudin Laurent , Broccardo Cyril , Peterson Katherine M. , Koch Christine , Arnould Isabelle , Prades Catherine , Duverger Nicholas , Funke Harald , Assman Gerd , Dinger Maria , Dean Michael , Chimini Giovanna , Santamarina-Fojo Silvia , Fredrickson Donald S. , Denefle Patrice , Brewer H. Bryan
Published: July 2002

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins.
ABC1
, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human
ABC1
gene and the identification of a mutation in the
ABC1
gene from the original Tangier disease kindred. The organization of the human
ABC1
gene is similar to that of the mouse
ABC1
gene and other related
ABC
genes. The
ABC1
gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the
ABC1
gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a
Mnl
1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human
ABC1
gene and identify a frameshift mutation in the
ABC1
gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the
ABC1
gene and the analysis of additional
ABC1
mutations in patients with Tangier disease.
Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and β-chemokine production
Authors: Gauduin Marie-Claire , Glickman Rhona L. , Ahmad Shabbir , Yilma Tilahun , Johnson R. Paul
Published: July 2002

Immunization with live attenuated simian immunodeficiency virus (SIV) strains has proved to be one of the most effective strategies to induce protective immunity in the SIV/macaque model. To better understand the role that CD4+T helper responses may play in mediating protection in this model, we characterized SIV-specific proliferative and cytokine responses in macaques immunized with live attenuated SIV strains. Macaques chronically infected with live attenuated SIV had strong proliferative responses to SIV proteins, with stimulation indices of up to 74. The magnitude of the proliferative response to SIV Gag varied inversely with the degree of attenuation; Gag-specific but not envelope-specific responses were lower in animals infected with more highly attenuated SIV strains. SIV-specific stimulation of lymphocytes from vaccinated macaques resulted in secretion of interferon-γ, IL-2, regulated-upon-activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β but not IL-4 or IL-10. Intracellular flow cytometric analysis documented that, in macaques vaccinated with SIVmac239Δnef, up to 2% of all CD4+T cells were specific for SIV p55. The ability of live attenuated SIV to induce a strong, sustained type 1 T helper response may play a role in the success of this vaccination approach to generate protection against challenge with wild-type SIV.