bluuebunny/crossref_metadata_2025 huggingface dataset:
Intermolecular thiol cross-linking via loops in the lactose permease of
Escherichia coli
Authors: Ermolova Natalia , Guan Lan , Kaback H. Ronald
Published: September 2003

Previous experiments using intermolecular thiol cross-linking to determine
surface-exposed positions in the transmembrane helices of the lactose permease
suggest that only positions accessible from the aqueous phase are susceptible
to cross-linking. This approach is now extended to most of the remaining
positions in the molecule. Of an additional 143 single-Cys mutants studied,
homodimer formation is observed with both a 5-Å- and a 21-Å-long
crosslinking agent containing
bis
-methane thiosulfonate reactive
groups in 33 mutants and exclusively with the 21-Å-long reagent in 43
mutants. Furthermore, intermolecular cross-linking has little or no effect on
transport activity, thereby providing further support for the argument that
lactose permease is functionally, as well as structurally, a monomer in the
membrane. In addition, evidence is presented indicating that reentrance loops
are unlikely in this polytopic membrane transport protein.
B7x: A widely expressed B7 family member that inhibits T cell activation
Authors: Zang Xingxing , Loke P'ng , Kim Jayon , Murphy Kenneth , Waitz Rebecca , Allison James P.
Published: September 2003

B7 family proteins provide costimulatory signals that regulate T cell
responses. Here we report the third set of B7 family-related T cell inhibitory
molecules with the identification of a homolog of the B7 family, B7x. It is
expressed in immune cells, nonlymphoid tissues, and some tumor cell lines. B7x
inhibits cell-cycle progression, proliferation, and cytokine production of
both CD4
+
and CD8
+
T cells. B7x binds a receptor that is
expressed on activated, but not resting T cells that is distinct from known
CD28 family members. Its receptor may be a recently identified inhibitory
molecule, B and T lymphocyte attenuator. These studies identify a
costimulatory pathway that may have a unique function in downregulation of
tissue-specific autoimmunity and antitumor responses.
The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA
Authors: Bermudez Vladimir P. , Maniwa Yoshimasa , Tappin Inger , Ozato Keiko , Yokomori Kyoko , Hurwitz Jerard
Published: September 2003

The linkage of sister chromatids after DNA replication ensures the faithful
inheritance of chromosomes by daughter cells. In budding yeast, the
establishment of sister chromatid cohesion requires Ctf8, Dcc1, and Ctf18, a
homologue of the p140 subunit of the replication factor C (RFC). In this
report we demonstrate that in 293T cells, Flag-tagged Ctf18 forms a
seven-subunit cohesion-RFC complex comprised of Ctf18, Dcc1, Ctf8, RFCp40,
RFCp38, RFCp37, and RFCp36 (Ctf18-RFC). We demonstrate that a stoichiometric
heteroheptameric Ctf18-RFC complex can be assembled by coexpressing the seven
proteins in baculovirus-infected insect cells. In addition, the two other
stable subcomplexes were formed, which include a pentameric complex comprised
of Ctf18, RFCp40, RFCp38, RFCp37, and RFCp36 and a dimeric Dcc1-Ctf8. Both the
five- and seven-subunit Ctf18-RFC complexes bind to single-stranded and primed
DNAs and possess weak ATPase activity that is stimulated by the addition of
primed DNA and proliferating cell nuclear antigen (PCNA). These complexes
catalyzed the ATP-dependent loading of PCNA onto primed and gapped DNA but not
onto double-stranded nicked or single-stranded circular DNAs. Consistent with
these observations, both Ctf18-RFC complexes substituted for the replicative
RFC in the PCNA-dependent DNA polymerase δ-catalyzed DNA replication
reaction. These results support a model in which sister chromatid cohesion is
linked to DNA replication.
Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-γ-induced class II transactivator
Authors: Cenci Simone , Toraldo Gianluca , Weitzmann M. Neale , Roggia Cristiana , Gao Yuhao , Qian Wei Ping , Sierra Oscar , Pacifici Roberto
Published: September 2003

Expansion of the pool of tumor necrosis factor (TNF)-α-producing T cells is instrumental for the bone loss induced by estrogen deficiency, but the responsible mechanism is unknown. Here we show that ovariectomy up-regulates IFN-γ-induced class II transactivator, a multitarget immune modulator, resulting in increased antigen presentation by macrophages, enhanced T cell activation, and prolonged lifespan of active T cells. Up-regulation of class II transactivator derives from increased production of IFN-γ by T helper 1 cells, resulting from enhanced secretion of IL-12 and IL-18 by macrophages. The resulting T cell expansion and bone loss are preventedin vivoby both blockade of antigen presenting cell-induced T cell activation, and silencing of IFN-γ receptor signaling. Thus, increased IFN-γ-induced class II transactivator expression and the resulting enhanced T cell proliferation and lifespan are critical to the bone wasting effect of estrogen deficiency.
Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1
Authors: Nimonkar Amitabh V. , Boehmer Paul E.
Published: September 2003

The repair of double-strand DNA breaks by homologous recombination is
essential for the maintenance of genome stability. In herpes simplex virus 1,
double-strand DNA breaks may arise as a consequence of replication fork
collapse at sites of oxidative damage, which is known to be induced upon viral
infection. Double-strand DNA breaks are also generated by cleavage of viral
a
sequences by endonuclease G during genome isomerization. We have
reconstituted a system using purified proteins in which strand invasion is
coupled with DNA synthesis. In this system, the viral single-strand
DNA-binding protein promotes assimilation of single-stranded DNA into a
homologous supercoiled plasmid, resulting in the formation of a displacement
loop. The 3′ terminus of the invading DNA serves as a primer for
long-chain DNA synthesis promoted by the viral DNA replication proteins,
including the polymerase and helicase-primase. Efficient extension of the
invading primer also requires a DNA-relaxing enzyme (eukaryotic topoisomerase
I or DNA gyrase). The viral polymerase by itself is insufficient for DNA
synthesis, and a DNA-relaxing enzyme cannot substitute for the viral
helicase-primase. The viral single-strand DNA-binding protein, in addition to
its role in the invasion process, is also required for long-chain DNA
synthesis. Form X, a topologically distinct, positively supercoiled form of
displacement-loop, does not serve as a template for DNA synthesis. These
observations support a model in which recombination and replication contribute
toward maintaining viral genomic stability by repairing double-strand breaks.
They also account for the extensive branching observed during viral
replication
in vivo
.
Development of object concepts in infancy: Evidence for early learning in an eye-tracking paradigm
Authors: Johnson Scott P. , Amso Dima , Slemmer Jonathan A.
Published: September 2003

Concepts of objects as enduring and complete across space and time have been documented in infants within several months after birth, but little is known about how such concepts arise during development. Current theories that stress innate knowledge may neglect the potential contributions of experience to guide acquisition of object concepts. To examine whether learning plays an important role in early development of object representations, we used an eye-tracking paradigm with 4- and 6-month-old infants who were provided with an initial period of experience viewing an unoccluded trajectory, or no experience with this particular stimulus. After exposure to the unoccluded trajectory for only 2 min, there was a reliable increase in 4-month-old infants' anticipatory eye movement when the infants subsequently viewed occluded-trajectory displays, relative to 4-month-old infants who did not receive this experience. This effect of training in 4-month-old infants was found to generalize to another category of trajectory orientation. Older infants received no additional benefit from training, most likely because they enter the task capable of forming robust object representations under these conditions. This finding provides compelling evidence that very brief training facilitated formation of object representations, and suggests more generally that infants learn such representations from real-world experience viewing objects undergoing occlusion and disocclusion.
Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphoma has a plasma cell gene expression profile
Authors: Jenner Richard G. , Maillard Karine , Cattini Nicola , Weiss Robin A. , Boshoff Chris , Wooster Richard , Kellam Paul
Published: September 2003

Kaposi's sarcoma-associated herpesvirus is associated with three human
tumors: Kaposi's sarcoma, and the B cell lymphomas, plasmablastic lymphoma
associated with multicentric Castleman's disease, and primary effusion
lymphoma (PEL). Epstein-Barr virus, the closest human relative of Kaposi's
sarcoma-associated herpesvirus, mimics host B cell signaling pathways to
direct B cell development toward a memory B cell phenotype. Epstein-Barr
virus-associated B cell tumors are presumed to arise as a consequence of this
virus-mediated B cell activation. The stage of B cell development represented
by PEL, how this stage relates to tumor pathology, and how this information
may be used to treat the disease are largely unknown. In this study we used
gene expression profiling to order a range of B cell tumors by stage of
development. PEL gene expression closely resembles that of malignant plasma
cells, including the low expression of mature B cell genes. The unfolded
protein response is partially activated in PEL, but is fully activated in
plasma cell tumors, linking endoplasmic reticulum stress to plasma cell
development through XBP-1. PEL cells can be defined by the overexpression of
genes involved in inflammation, cell adhesion, and invasion, which may be
responsible for their presentation in body cavities. Similar to malignant
plasma cells, all PEL samples tested express the vitamin D receptor and are
sensitive to the vitamin D analogue drug EB 1089 (Seocalcitol).
Manganese activation of superoxide dismutase 2 in
Saccharomyces cerevisiae
requires
MTM1
, a member of the mitochondrial carrier family
Authors: Luk Edward , Carroll Mark , Baker Michelle , Culotta Valeria Cizewski
Published: September 2003

Manganese-containing superoxide dismutase (SOD2) plays a critical role in
guarding against mitochondrial oxidative stress and is essential for survival
of many organisms. Despite the recognized importance of SOD2, nothing is known
regarding the mechanisms by which this nuclear-encoded protein is converted to
an active enzyme in the mitochondrial matrix. To search for factors that
participate in the posttranslational activation of SOD2, we screened for yeast
genes that when mutated lead to SOD2 inactivation and identified a single ORF,
YGR257c
. The encoded protein localizes to the mitochondria and
represents a member of the yeast mitochondrial carrier family.
YGR257c
was previously recognized as the homologue to human CGI-69, a
widely expressed mitochondrial carrier family of unknown function. Our studies
suggest a connection with SOD2, and we have named the yeast gene
MTM1
for manganese trafficking factor for mitochondrial SOD2. Inactivation of yeast
MTM1
leads to loss of SOD2 activity that is restored only when cells
are treated with high supplements of manganese, but not other heavy metals,
indicative of manganese deficiency in the SOD2 polypeptide. Surprisingly, the
mitochondrial organelle of
mtm1
Δ mutants shows no deficiency in
manganese levels. Moreover,
mtm1
Δ mutations do not impair
activity of a cytosolic version of manganese SOD. We propose that Mtm1p
functions in the mitochondrial activation of SOD2 by specifically facilitating
insertion of the essential manganese cofactor.
Osmotic water transport through carbon nanotube membranes
Authors: Kalra Amrit , Garde Shekhar , Hummer Gerhard
Published: September 2003

We use molecular dynamics simulations to study osmotically driven transport of water molecules through hexagonally packed carbon nanotube membranes. Our simulation setup comprises two such semipermeable membranes separating compartments of pure water and salt solution. The osmotic force drives water flow from the pure-water to the salt-solution compartment. Monitoring the flow at molecular resolution reveals several distinct features of nanoscale flows. In particular, thermal fluctuations become significant at the nanoscopic length scales, and as a result, the flow is stochastic in nature. Further, the flow appears frictionless and is limited primarily by the barriers at the entry and exit of the nanotube pore. The observed flow rates are high (5.8 water molecules per nanosecond and nanotube), comparable to those through the transmembrane protein aquaporin-1, and are practically independent of the length of the nanotube, in contrast to predictions of macroscopic hydrodynamics. All of these distinct characteristics of nanoscopic water flow can be modeled quantitatively by a 1D continuous-time random walk. At long times, the pure-water compartment is drained, and the net flow of water is interrupted by the formation of structured solvation layers of water sandwiched between two nanotube membranes. Structural and thermodynamic aspects of confined water monolayers are studied.
Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants
Authors: Schmelz Eric A. , Engelberth Juergen , Alborn Hans T. , O'Donnell Phillip , Sammons Matt , Toshima Hiroaki , Tumlinson James H.
Published: September 2003

Phytohormones regulate the protective responses of plants against both
biotic and abiotic stresses by means of synergistic or antagonistic actions
referred to as signaling crosstalk. A bottleneck in crosstalk research is the
quantification of numerous interacting phytohormones and regulators. The
chemical analysis of salicylic acid, jasmonic acid, indole-3-acetic acid, and
abscisic acid is typically achieved by using separate and complex
methodologies. Moreover, pathogen-produced phytohormone mimics, such as the
phytotoxin coronatine (COR), have not been directly quantified in plant
tissues. We address these problems by using a simple preparation and a
GC-MS-based metabolic profiling approach. Plant tissue is extracted in aqueous
1-propanol and mixed with dichloromethane. Carboxylic acids present in the
organic layer are methylated by using trimethylsilyldiazomethane; analytes are
volatilized under heat, collected on a polymeric absorbent, and eluted with
solvent into a sample vial. Analytes are separated by using gas chromatography
and quantified by using chemical-ionization mass spectrometry that produces
predominantly [M+H]
+
parent ions. We use this technique to examine
levels of COR, phytohormones, and volatile organic compounds in model systems,
including
Arabidopsis thaliana
during infection with
Pseudomonas
syringae
pv.
tomato
DC3000, corn (
Zea mays
) under
herbivory by corn earworm (
Helicoverpa zea
), tobacco (
Nicotiana
tabacum
) after mechanical damage, and tomato (
Lycopersicon
esculentum
) during drought stress. Numerous complex changes induced by
pathogen infection, including the accumulation of COR, salicylic acid,
jasmonic acid, indole-3-acetic acid, and abscisic acid illustrate the
potential and simplicity of this approach in quantifying signaling crosstalk
interactions that occur at the level of synthesis and accumulation.
Modulation of DNA damage-induced apoptosis by cell adhesion is independently mediated by p53 and c-Abl
Authors: Truong Tony , Sun Guizhen , Doorly Michael , Wang Jean Y. J. , Schwartz Martin Alexander
Published: September 2003

Conventional cancer therapies are based on preferential killing of tumor
cells by DNA damage. Previous work showed that, for certain cell types, loss
of integrin-mediated adhesion decreased the apoptotic response to DNA damage
because of decreased p53 levels after detachment from the extracellular
matrix. Integrin ligation restored p53 and sensitivity to DNA damage. In this
study, we show that c-Abl mediates a second pathway by which adhesion to
extracellular matrix regulates cell killing by chemotherapeutic agents
5-arabinofuranosylcytosine, cisplatin, and camptothecin. Activation of c-Abl
tyrosine kinase by DNA damage requires cell adhesion. Abl-dependent
stabilization of p73, a p53-related proapoptotic transcription factor, is also
adhesion-dependent. Sensitivity to the Abl inhibitor STI571 suggests
differential utilization of the p53 and c-Abl/p73 pathways by different tumor
cell lines. These data suggest that killing of p53-negative tumor cells by
chemotherapy would be enhanced by integrin ligation to activate the
alternative c-Abl/p73 pathway.
Biomimetic organization: Octapeptide self-assembly into nanotubes of viral capsid-like dimension
Authors: Valéry Céline , Paternostre Maïté , Robert Bruno , Gulik-Krzywicki Thaddée , Narayanan Theyencheri , Dedieu Jean-Claude , Keller Gérard , Torres Maria-Luisa , Cherif-Cheikh Roland , Calvo Pilar , Artzner Franck
Published: September 2003

The controlled self-assembly of complex molecules into well defined hierarchical structures is a promising route for fabricating nanostructures. These nanoscale structures can be realized by naturally occurring proteins such as tobacco mosaic virus, capsid proteins, tubulin, actin, etc. Here, we report a simple alternative method based on self-assembling nanotubes formed by a synthetic therapeutic octapeptide, Lanreotide in water. We used a multidisciplinary approach involving optical and electron microscopies, vibrational spectroscopies, and small and wide angle x-ray scattering to elucidate the hierarchy of structures exhibited by this system. The results revealed the hexagonal packing of nanotubes, and high degree of monodispersity in the tube diameter (244 Å) and wall thickness (≈18 Å). Moreover, the diameter is tunable by suitable modifications in the molecular structure. The self-assembly of the nanotubes occurs through the association of β-sheets driven by amphiphilicity and a systematic aromatic/aliphatic side chain segregation. This original and simple system is a unique example for the study of complex self-assembling processes generated byde novomolecules or amyloid peptides.
Allopatric genetic origins for sympatric host-plant shifts and race formation in
Rhagoletis
Authors: Feder Jeffrey L. , Berlocher Stewart H. , Roethele Joseph B. , Dambroski Hattie , Smith James J. , Perry William L. , Gavrilovic Vesna , Filchak Kenneth E. , Rull Juan , Aluja Martin
Published: September 2003

Tephritid fruit flies belonging to the
Rhagoletis pomonella
sibling species complex are controversial because they have been proposed to
diverge in sympatry (in the absence of geographic isolation) by shifting and
adapting to new host plants. Here, we report evidence suggesting a surprising
source of genetic variation contributing to sympatric host shifts for these
flies. From DNA sequence data for three nuclear loci and mtDNA, we infer that
an ancestral, hawthorn-infesting
R. pomonella
population became
geographically subdivided into Mexican and North American isolates ≈1.57
million years ago. Episodes of gene flow from Mexico subsequently infused the
North American population with inversion polymorphism affecting key diapause
traits, forming adaptive clines. Sometime later (perhaps ±1 million
years), diapause variation in the latitudinal clines appears to have aided
North American flies in adapting to a variety of plants with differing
fruiting times, helping to spawn several new taxa. Thus, important raw genetic
material facilitating the adaptive radiation of
R. pomonella
originated in a different time and place than the proximate ecological host
shifts triggering sympatric divergence.
Genetic footprints of demographic expansion in North America, but not Amazonia, during the Late Quaternary
Authors: Lessa Enrique P. , Cook Joseph A. , Patton James L.
Published: September 2003

The biotic consequences of climate change have attracted considerable
attention. In particular, the “refugial debate” centers on the
possible retraction of habitats to limited areas that may have served as
refuges for many associated species, especially during glaciations of the
Quaternary. One prediction of such scenarios is that populations must have
experienced substantial growth accompanying climatic amelioration and the
occupation of newly expanded habitats. We used coalescence theory to examine
the genetic evidence, or lack thereof, for late Pleistocene refugia of boreal
North American and tropical Amazonian mammals. We found substantial and
concordant evidence of demographic expansion in North American mammals,
particularly at higher latitudes. In contrast, small mammals from western
Amazonia appear to have experienced limited or no demographic expansion after
the Late Pleistocene. Thus, demographic responses to climate change can be
tracked genetically and appear to vary substantially across the latitudinal
gradient of biotic diversity.
Group interest versus self-interest in smallpox vaccination policy
Authors: Bauch Chris T. , Galvani Alison P. , Earn David J. D.
Published: September 2003

The recent threat of bioterrorism has fueled debate on smallpox vaccination policy for the United States. Certain policy proposals call for voluntary mass vaccination; however, if individuals decide whether to vaccinate according to self-interest, the level of herd immunity achieved may differ from what is best for the population as a whole. We present a synthesis of game theory and epidemic modeling that formalizes this conflict between self-interest and group interest and shows that voluntary vaccination is unlikely to reach the group-optimal level. This shortfall results in a substantial increase in expected mortality after an attack.
γ-Tubulin function during female germ-cell development and oogenesis in
Drosophila
Authors: Tavosanis Gaia , Gonzalez Cayetano
Published: September 2003

A series of unconventional microtubule organizing centers play a
fundamental role during egg chamber development in
Drosophila
. To
gain a better understanding of their molecular nature, we have studied the
centrosomal component γ-tubulin during
Drosophila
oogenesis. We
find that although single mutations in either of the two γ-tubulin genes
identified in
Drosophila
do not affect oogenesis progression the
simultaneous depletion of both protein products has severe consequences. The
combination of loss-of-function mutant alleles for the two γ-tubulin
genes leads to mitotic defects in female germ cells, resulting in agametic
ovaries. A combination of weaker mutant alleles instead allows female
germ-cell development to proceed, although the resulting egg chambers display
pleiotropic abnormalities, most frequently affecting the number of nurse cells
and oocytes per egg chamber. Thus, γ-tubulin is required for several
processes at different stages of germ-cell development and oogenesis,
including oocyte determination and differentiation. Our data provide a
functional link between a component of the peri-centriolar material, such as
γ-tubulin, and microtubule organization during
Drosophila
oogenesis. In addition, our results show that γ-tubulin is required for
female germcell proliferation and that the two γ-tubulins present in
Drosophila
are functionally equivalent during female germ-cell
development and oogenesis.
The complete genome sequence of the
Arabidopsis
and tomato pathogen
Pseudomonas syringae
pv.
tomato
DC3000
Authors: Buell C. Robin , Joardar Vinita , Lindeberg Magdalen , Selengut Jeremy , Paulsen Ian T. , Gwinn Michelle L. , Dodson Robert J. , Deboy Robert T. , Durkin A. Scott , Kolonay James F. , Madupu Ramana , Daugherty Sean , Brinkac Lauren , Beanan Maureen J. , Haft Daniel H. , Nelson William C. , Davidsen Tanja , Zafar Nikhat , Zhou Liwei , Liu Jia , Yuan Qiaoping , Khouri Hoda , Fedorova Nadia , Tran Bao , Russell Daniel , Berry Kristi , Utterback Teresa , Van Aken Susan E. , Feldblyum Tamara V. , D'Ascenzo Mark , Deng Wen-Ling , Ramos Adela R. , Alfano James R. , Cartinhour Samuel , Chatterjee Arun K. , Delaney Terrence P. , Lazarowitz Sondra G. , Martin Gregory B. , Schneider David J. , Tang Xiaoyan , Bender Carol L. , White Owen , Fraser Claire M. , Collmer Alan
Published: September 2003

We report the complete genome sequence of the model bacterial pathogen
Pseudomonas syringae
pathovar
tomato
DC3000 (DC3000), which
is pathogenic on tomato and
Arabidopsis thaliana
. The DC3000 genome
(6.5 megabases) contains a circular chromosome and two plasmids, which
collectively encode 5,763 ORFs. We identified 298 established and putative
virulence genes, including several clusters of genes encoding 31 confirmed and
19 predicted type III secretion system effector proteins. Many of the
virulence genes were members of paralogous families and also were proximal to
mobile elements, which collectively comprise 7% of the DC3000 genome. The
bacterium possesses a large repertoire of transporters for the acquisition of
nutrients, particularly sugars, as well as genes implicated in attachment to
plant surfaces. Over 12% of the genes are dedicated to regulation, which may
reflect the need for rapid adaptation to the diverse environments encountered
during epiphytic growth and pathogenesis. Comparative analyses confirmed a
high degree of similarity with two sequenced pseudomonads,
Pseudomonas
putida
and
Pseudomonas aeruginosa
, yet revealed 1,159 genes
unique to DC3000, of which 811 lack a known function.
Wilderness and biodiversity conservation
Authors: Mittermeier R. A. , Mittermeier C. G. , Brooks T. M. , Pilgrim J. D. , Konstant W. R. , da Fonseca G. A. B. , Kormos C.
Published: September 2003

Human pressure threatens many species and ecosystems, so conservation
efforts necessarily prioritize saving them. However, conservation should
clearly be proactive wherever possible. In this article, we assess the
biodiversity conservation value, and specifically the irreplaceability in
terms of species endemism, of those of the planet's ecosystems that remain
intact. We find that 24 wilderness areas, all > 1 million hectares, are
> 70% intact and have human densities of less than or equal to five people
per km
2
. This wilderness covers 44% of all land but is inhabited by
only 3% of people. Given this sparse population, wilderness conservation is
cost-effective, especially if ecosystem service value is incorporated.
Soberingly, however, most wilderness is not speciose: only 18% of plants and
10% of terrestrial vertebrates are endemic to individual wildernesses, the
majority restricted to Amazonia, Congo, New Guinea, the Miombo-Mopane
woodlands, and the North American deserts. Global conservation strategy must
target these five wildernesses while continuing to prioritize threatened
biodiversity hotspots.
Niemann-Pick C heterozygosity confers resistance to lesional necrosis and macrophage apoptosis in murine atherosclerosis
Authors: Feng Bo , Zhang Dajun , Kuriakose George , Devlin Cecillia M. , Kockx Mark , Tabas Ira
Published: September 2003

Macrophage death in advanced atherosclerotic lesions leads to lesional
necrosis and likely promotes plaque instability, a precursor of acute vascular
events. Macrophages in advanced lesions accumulate large amounts of
unesterified cholesterol, which is a potent inducer of macrophage apoptosis.
We have shown recently that induction of apoptosis in cultured macrophages
requires cholesterol trafficking to the endoplasmic reticulum (ER). Moreover,
macrophages from mice with a heterozygous mutation in the
cholesterol-trafficking protein Npc1 have a selective defect in cholesterol
trafficking to the ER and are protected from cholesterol-induced apoptosis.
The goal of the present study was to test the importance of intracellular
cholesterol trafficking in atherosclerotic lesional macrophage death by
comparing lesion morphology in
Npc1
+/+
;
Apoe
-/-
and
Npc1
+/-
;
Apoe
-/-
mice. Although
advanced lesions in
Npc1
+/+
;
Apoe
-/-
mice had extensive acellular areas that were rich in unesterified cholesterol
and macrophage debris, the lesions of
Npc1
+/-
;
Apoe
-/-
mice were
substantially more cellular and less necrotic. Moreover, compared with
Npc1
+/-
;
Apoe
-/-
lesions,
Npc1
+/+
;
Apoe
-/-
lesions had a greater
number of large, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP
nick end labeling)-positive areas surrounding necrotic areas, indicative of
macrophage apoptosis. These differences were observed despite similar total
lesion area and similar plasma lipid levels in the two groups of mice. These
data provide
in vivo
evidence that intact intracellular cholesterol
trafficking is important for macrophage apoptosis in advanced atherosclerotic
lesions and that the ER-based model of cholesterol-induced cytotoxicity is
physiologically relevant. Moreover, by showing that lesional necrosis can be
diminished by a subtle defect in intracellular trafficking, these findings
suggest therapeutic strategies to stabilize atherosclerotic plaques.
Comparing the continuous representation of time-series expression profiles to identify differentially expressed genes
Authors: Bar-Joseph Ziv , Gerber Georg , Simon Itamar , Gifford David K. , Jaakkola Tommi S.
Published: September 2003

We present a general algorithm to detect genes differentially expressed between two nonhomogeneous time-series data sets. As increasing amounts of high-throughput biological data become available, a major challenge in genomic and computational biology is to develop methods for comparing data from different experimental sources. Time-series whole-genome expression data are a particularly valuable source of information because they can describe an unfolding biological process such as the cell cycle or immune response. However, comparisons of time-series expression data sets are hindered by biological and experimental inconsistencies such as differences in sampling rate, variations in the timing of biological processes, and the lack of repeats. Our algorithm overcomes these difficulties by using a continuous representation for time-series data and combining a noise model for individual samples with a global difference measure. We introduce a corresponding statistical method for computing the significance of this differential expression measure. We used our algorithm to compare cell-cycle-dependent gene expression in wild-type and knockout yeast strains. Our algorithm identified a set of 56 differentially expressed genes, and these results were validated by using independent protein-DNA-binding data. Unlike previous methods, our algorithm was also able to identify 22 non-cell-cycle-regulated genes as differentially expressed. This set of genes is significantly correlated in a set of independent expression experiments, suggesting additional roles for the transcription factors Fkh1 and Fkh2 in controlling cellular activity in yeast.
Breast cancer classification and prognosis based on gene expression profiles from a population-based study
Authors: Sotiriou Christos , Neo Soek-Ying , McShane Lisa M. , Korn Edward L. , Long Philip M. , Jazaeri Amir , Martiat Philippe , Fox Steve B. , Harris Adrian L. , Liu Edison T.
Published: September 2003

Comprehensive gene expression patterns generated from cDNA microarrays were
correlated with detailed clinico-pathological characteristics and clinical
outcome in an unselected group of 99 node-negative and node-positive breast
cancer patients. Gene expression patterns were found to be strongly associated
with estrogen receptor (ER) status and moderately associated with grade, but
not associated with menopausal status, nodal status, or tumor size.
Hierarchical cluster analysis segregated the tumors into two main groups based
on their ER status, which correlated well with basal and luminal
characteristics. Cox proportional hazards regression analysis identified 16
genes that were significantly associated with relapse-free survival at a
stringent significance level of 0.001 to account for multiple comparisons. Of
231 genes previously reported by others [van't Veer, L. J.,
et al.
(2002)
Nature
415, 530-536] as being associated with survival, 93
probe elements overlapped with the set of 7,650 probe elements represented on
the arrays used in this study. Hierarchical cluster analysis based on the set
of 93 probe elements segregated our population into two distinct subgroups
with different relapse-free survival (
P
< 0.03). The number of
these 93 probe elements showing significant univariate association with
relapse-free survival (
P
< 0.05) in the present study was 14,
representing 11 unique genes. Genes involved in cell cycle, DNA replication,
and chromosomal stability were consistently elevated in the various poor
prognostic groups. In addition, glutathione
S
-transferase M3 emerged
as an important survival marker in both studies. When taken together with
other array studies, our results highlight the consistent biological and
clinical associations with gene expression profiles.
Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR
Authors: Ardenkjær-Larsen Jan H. , Fridlund Björn , Gram Andreas , Hansson Georg , Hansson Lennart , Lerche Mathilde H. , Servin Rolf , Thaning Mikkel , Golman Klaes
Published: September 2003

A method for obtaining strongly polarized nuclear spins in solution has
been developed. The method uses low temperature, high magnetic field, and
dynamic nuclear polarization (DNP) to strongly polarize nuclear spins in the
solid state. The solid sample is subsequently dissolved rapidly in a suitable
solvent to create a solution of molecules with hyperpolarized nuclear spins.
The polarization is performed in a DNP polarizer, consisting of a
super-conducting magnet (3.35 T) and a liquid-helium cooled sample space. The
sample is irradiated with microwaves at ≈94 GHz. Subsequent to
polarization, the sample is dissolved by an injection system inside the DNP
magnet. The dissolution process effectively preserves the nuclear
polarization. The resulting hyperpolarized liquid sample can be transferred to
a high-resolution NMR spectrometer, where an enhanced NMR signal can be
acquired, or it may be used as an agent for
in vivo
imaging or
spectroscopy. In this article we describe the use of the method on aqueous
solutions of [
13
C]urea. Polarizations of 37% for
13
C and
7.8% for
15
N, respectively, were obtained after the dissolution.
These polarizations correspond to an enhancement of 44,400 for
13
C
and 23,500 for
15
N, respectively, compared with thermal equilibrium
at 9.4 T and room temperature. The method can be used generally for signal
enhancement and reduction of measurement time in liquid-state NMR and opens up
for a variety of
in vitro
and
in vivo
applications of
DNP-enhanced NMR.
Molecular imaging with endogenous substances
Authors: Golman Klaes , Ardenkjær-Larsen Jan H. , Petersson J. Stefan , Månsson Sven , Leunbach Ib
Published: September 2003

Dynamic nuclear polarization has enabled hyperpolarization of nuclei such
as
13
C and
15
N in endogenous substances. The resulting
high nuclear polarization makes it possible to perform subsecond
13
C MRI. By using the dynamic nuclear polarization
hyperpolarization technique, 10% polarization was obtained in an aqueous
solution of 100 mM
13
C-labeled urea, ready for injection. The
in vivo T
1
relaxation time of
13
C in the urea
solution was determined to 20 ± 2 s. Due to the long relaxation time,
it is possible to use the hyperpolarized substance for medical imaging. A
series of high-resolution (≈1-mm) magnetic resonance images were acquired,
each with a scan time of 240 ms, 0-5 s after an i.v. injection of the
hyperpolarized aqueous [
13
C]urea solution in a rat. The results
show that it is possible to perform
13
C angiography with a
signal-to-noise ratio of ≈275 in ≈0.25 s. Perfusion studies with
endogenous substances may allow higher spatial and/or temporal resolution than
is possible with current proton imaging techniques.
Effectiveness of diabetes mellitus screening recommendations
Authors: Dallo Florence J. , Weller Susan C.
Published: September 2003

Screening guidelines proposed by the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus have been endorsed by several medical societies. However, one-third of cases are undiagnosed, and complications at the time of diagnosis indicate that disease may have been present for several years before diagnosis. This study evaluates the effectiveness of the guidelines for detecting new cases of diabetes mellitus. By using a cross-sectional, representative sample of the United States (National Health and Nutritional Examination Survey, NHANES III), the guidelines are tested on adults, 20 years and older without a prior diagnosis of diabetes. Individuals are classified as nondiabetics (
n
= 6,241) or as having undiagnosed diabetes (
n
= 274) based on their blood glucose. Screening when one risk factor is present, as stated in the guidelines, has a true-positive rate of 100% and would require that 83% of the population be tested. Screening when two risk factors are present is more efficient, with a comparable true-positive rate (98%), but requires that only 59% of the population be tested. A notable finding is the earlier age of onset among minorities, which may be associated with other health disparities. Because diabetes occurs at younger ages in minorities, screening whites who are ≥40 and minorities ≥30 years of age has a high true-positive rate (95%) and also reduces testing (60%). The screening guidelines would be effective, if followed, and would essentially eliminate undiagnosed cases of diabetes.
Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets
Authors: Abu-Elheiga Lutfi , Oh Wonkeun , Kordari Parichher , Wakil Salih J.
Published: September 2003

Malonyl-CoA, generated by acetyl-CoA carboxylases ACC1 and ACC2, is a key
metabolite in the control of fatty acid synthesis and oxidation in response to
dietary changes. ACC2 is associated to the mitochondria, and
Acc2
-/-
mice have a normal lifespan and higher fatty acid
oxidation rate and accumulate less fat. Mutant mice fed
high-fat/high-carbohydrate diets weighed less than their WT cohorts,
accumulated less fat, and maintained normal levels of insulin and glucose,
whereas the WT mice became type-2 diabetic with hyperglycemic and
hyperinsulinemic status. Fatty acid oxidation rates in the soleus muscle and
in hepatocytes of
Acc2
-/-
mice were significantly higher
than those of WT cohorts and were not affected by the addition of insulin.
mRNA levels of uncoupling proteins (UCPs) were significantly higher in
adipose, heart (UCP2), and muscle (UCP3) tissues of mutant mice compared with
those of the WT. The increase in the UCP levels along with increased fatty
acid oxidation may play an essential role in the regulation of energy
expenditure. Lowering intracellular fatty acid accumulation in the mutant
relative to that of the WT mice may thus impact glucose transport by higher
GLUT4 activity and insulin sensitivity. These results suggest that ACC2 plays
an essential role in controlling fatty acid oxidation and is a potential
target in therapy against obesity and related diseases.
A noncanonical sequence phosphorylated by casein kinase 1 in β-catenin may play a role in casein kinase 1 targeting of important signaling proteins
Authors: Marin Oriano , Bustos Victor H. , Cesaro Luca , Meggio Flavio , Pagano Mario A. , Antonelli Marcelo , Allende Catherine C. , Pinna Lorenzo A. , Allende Jorge E.
Published: September 2003

Protein kinase casein kinase 1 (CK1) phosphorylates Ser-45 of
β-catenin, “priming” the subsequent phosphorylation by
glycogen synthase-3 of residues 41, 37, and 33. This concerted phosphorylation
of β-catenin signals its degradation and prevents its function in
triggering cell division. The sequence around Ser-45 does not conform to the
canonical consensus for CK1 substrates, which prescribes either phosphoamino
acids or acidic residues in position n-3 from the target serine. However, the
β-catenin sequence downstream from Ser-45 is very similar to a sequence
recognized by CK1 in nuclear factor for activated T cells 4. The common
features include an SLS motif followed two to five residues downstream by a
cluster of acidic residues. Synthetic peptides reproducing residues 38-65 of
β-catenin were assayed with purified rat liver CK1 or recombinant
CK1α and CK1αL from zebrafish. The results demonstrate that SLS
and acidic cluster motifs are crucial for CK1 recognition. Pro-44 and Pro-52
are also important for efficient phosphorylation. Similar results were
obtained with the different isoforms of CK1. Phosphorylation of mutants of
full-length recombinant β-catenin from zebrafish confirmed the importance
of the SLS and acidic cluster motifs. A search for proteins with similar
motifs yielded, among other proteins, adenomatous polyposis coli, previously
found to be phosphorylated by CK1. There is a strong correlation of
β-catenin mutations found in thyroid tumors with the motifs recognized by
CK1 in this protein.
Brain potentials associated with conscious aftereffects induced by unseen stimuli in a blindsight subject
Authors: Weiskrantz L. , Rao A. , Hodinott-Hill I. , Nobre A. C. , Cowey A.
Published: September 2003

The study is of brain activity in a blindsight subject (D.B.), who reports
conscious visual afterimages of stimuli of which he is unaware when they are
presented. This contrast offered a unique opportunity to study event-related
potential recordings of conscious versus unconscious visual phenomena
generated by the very same stimulus in the identical locus of the visual
field. The behavioral results confirmed the reliability of the difference in
the subject's report for inducing stimuli versus their aftereffects. The
rationale of the event-related potential analysis was to subtract
“on” signals from “off” signals, the latter associated
with the onset of conscious events and the former for events that remained
unconscious. Because there are inherent differences in on and off potentials,
the subtractive resultants for the blind hemifield were compared with the same
subtractions for the good hemifield when the subject was aware both of the
stimuli and their afterimages. A differential pattern in subtractive
resultants emerged with a strong anterior left frontal focus for the blind
field and a posterior focus for the intact field. The results are compared
with other studies suggesting an anterior focus for conscious visual
events.
Honor thy symbionts
Authors: Xu Jian , Gordon Jeffrey I.
Published: September 2003

Our intestine is the site of an extraordinarily complex and dynamic environmentally transmitted consortial symbiosis. The molecular foundations of beneficial symbiotic host-bacterial relationships in the gut are being revealed in part from studies of simplified models of this ecosystem, where germ-free mice are colonized with specified members of the microbial community, and in part from comparisons of the genomes of members of the intestinal microbiota. The results emphasize the contributions of symbionts to postnatal gut development and host physiology, as well as the remarkable strategies these microorganisms have evolved to sustain their alliances. These points are illustrated by the human-Bacteroides thetaiotaomicronsymbiosis. Interdisciplinary studies of the effects of the intestinal environment on genome structure and function should provide important new insights about how microbes and humans have coevolved mutually beneficial relationships and new perspectives about the foundations of our health.
Unsuspected role of the brain morphogenetic geneOtx1in hematopoiesis
Authors: Levantini Elena , Giorgetti Alessandra , Cerisoli Francesco , Traggiai Elisabetta , Guidi Alessandra , Martin Richard , Acampora Dario , Aplan Peter D. , Keller Gordon , Simeone Antonio , Iscove Norman N. , Hoang Trang , Magli Maria Cristina
Published: September 2003

Otx1belongs to the paired class of homeobox genes and plays a pivotal role in brain development. Here, we show thatOtx1is expressed in hematopoietic pluripotent and erythroid progenitor cells. Moreover, bone marrow cells from mice lackingOtx1exhibit a cell-autonomous impairment of the erythroid compartment. In agreement with these results, molecular analysis revealed decreased levels of erythroid genes that include theSCLandGATA-1transcription factors. Accordingly, a gain of function of SCL rescues the erythroid deficiency inOtx1-/-mice. Taken together, our findings indicate a function forOtx1in the regulation of blood cell production.
A calmodulin-binding protein fromArabidopsishas an essential role in pollen germination
Authors: Golovkin Maxim , Reddy Anireddy S. N.
Published: September 2003

Calmodulin (CaM), a ubiquitous multifunctional calcium sensor in all eukaryotes, mediates calcium action by regulating the activity/function of many unrelated proteins. Although calcium and CaM are known to play a crucial role in pollen germination and pollen tube growth, the proteins that mediate their action have not been identified. We isolated three closely related CaM-binding proteins (NPG1, NPGR1, and NPGR2) fromArabidopsis. NPG1(No Pollen Germination1) is expressed only in pollen, whereas theNPG-related proteins (NPGR1andNPGR2) are expressed in pollen and other tissues. The bacterially expressed NPG1 bound three isoforms ofArabidopsisCaM in a calcium-dependent manner. To analyze the function ofNPG1, we performed a reverse genetics screen and isolated a mutant in whichNPG1is disrupted by a T-DNA insertion. Segregation and molecular analyses of theNPG1knockout mutant and a cross with a male sterile mutant indicate that the mutatedNPG1is not transmitted through the male gametophyte. Expression ofNPG1in the knockout mutant complemented the mutant phenotype. Analysis of pollen development in the knockout mutant by light microscopy showed normal pollen development. Pollen fromNPG1mutant in thequartetbackground has confirmed thatNPG1is dispensable for pollen development. However, germination studies with pollen from the mutant in thequartetbackground indicate that pollen carrying a mutant allele does not germinate. Our genetic, histological, and pollen germination studies with the knockout mutant line indicate thatNPG1is not necessary for microsporogenesis and gametogenesis but is essential for pollen germination.
WNT7a induces E-cadherin in lung cancer cells
Authors: Ohira Tatsuo , Gemmill Robert M. , Ferguson Kevin , Kusy Sophie , Roche Joëlle , Brambilla Elisabeth , Zeng Chan , Baron Anna , Bemis Lynne , Erickson Paul , Wilder Elizabeth , Rustgi Anil , Kitajewski Jan , Gabrielson Edward , Bremnes Roy , Franklin Wilbur , Drabkin Harry A.
Published: September 2003

E-cadherin loss in cancer is associated with de-differentiation, invasion,
and metastasis.
Drosophila
DE-cadherin is regulated by
Wnt/β-catenin signaling, although this has not been demonstrated in
mammalian cells. We previously reported that expression of WNT7a, encoded on
3p25, was frequently downregulated in lung cancer, and that loss of E-cadherin
or β-catenin was a poor prognostic feature. Here we show that WNT7a both
activates E-cadherin expression via a β-catenin specific mechanism in
lung cancer cells and is involved in a positive feedback loop. Li
+
,
a GSK3β inhibitor, led to E-cadherin induction in an inositol-independent
manner. Similarly, exposure to mWNT7a specifically induced free β-catenin
and E-cadherin. Among known transcriptional suppressors of E-cadherin, ZEB1
was uniquely correlated with E-cadherin loss in lung cancer cell lines, and
its inhibition by RNA interference resulted in E-cadherin induction.
Pharmacologic reversal of E-cadherin and WNT7a losses was achieved with
Li
+
, histone deacetylase inhibition, or in some cases only with
combined inhibitors. Our findings provide support that E-cadherin induction by
WNT/β-catenin signaling is an evolutionarily conserved pathway operative
in lung cancer cells, and that loss of WNT7a expression may be important in
lung cancer development or progression by its effects on E-cadherin.