bluuebunny/crossref_metadata_2025 huggingface dataset:
Statistical analysis of MPSS measurements: Application to the study of LPS-activated macrophage gene expression
Authors: Stolovitzky G. A. , Kundaje A. , Held G. A. , Duggar K. H. , Haudenschild C. D. , Zhou D. , Vasicek T. J. , Smith K. D. , Aderem A. , Roach J. C.
Published: January 2005

Massively Parallel Signature Sequencing (MPSS), a recently developed high-throughput transcription profiling technology, has the ability to profile almost every transcript in a sample without requiring prior knowledge of the sequence of the transcribed genes. As is the case with DNA microarrays, effective data analysis depends crucially on understanding how noise affects measurements. We analyze the sources of noise in MPSS and present a quantitative model describing the variability between replicate MPSS assays. We use this model to construct statistical hypotheses that test whether an observed change in gene expression in a pair-wise comparison is significant. This analysis is then extended to the determination of the significance of changes in expression levels measured over the course of a time series of measurements. We apply these analytic techniques to the study of a time series of MPSS gene expression measurements on LPS-stimulated macrophages. To evaluate our statistical significance metrics, we compare our results with published data on macrophage activation measured by using Affymetrix GeneChips.
α-Glucan, water dikinase (GWD): A plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity
Authors: Mikkelsen René , Mutenda Kudzai E. , Mant Alexandra , Schürmann Peter , Blennow Andreas
Published: January 2005

The recently discovered potato tuber (
Solanum tuberosum
) α-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the β-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx)
f
and Trx
m
from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx
f
being the more efficient, yielding an S
0.5
value of 0.4 μM. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.
Requirement of phospholipase D1 activity in H-Ras
V12
-induced transformation
Authors: Buchanan F. Gregory , McReynolds Matt , Couvillon Anthony , Kam Yoonseok , Holla Vijaykumar R. , DuBois Raymond N. , Exton John H.
Published: January 2005

The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H-Ras
V12
oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H-Ras
V12
oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.
Gating of acetylcholine receptor channels: Brownian motion across a broad transition state
Authors: Auerbach Anthony
Published: January 2005

Acetylcholine receptor channels (AChRs) are proteins that switch between stable “closed” and “open” conformations. In patch clamp recordings, diliganded AChR gating appears to be a simple, two-state reaction. However, mutagenesis studies indicate that during gating dozens of residues across the protein move asynchronously and are organized into rigid body gating domains (“blocks”). Moreover, there is an upper limit to the apparent channel opening rate constant. These observations suggest that the gating reaction has a broad, corrugated transition state region, with the maximum opening rate reflecting, in part, the mean first-passage time across this ensemble. Simulations reveal that a flat, isotropic energy profile for the transition state can account for many of the essential features of AChR gating. With this mechanism, concerted, local structural transitions that occur on the broad transition state ensemble give rise to fractional measures of reaction progress (Φ values) determined by rate-equilibrium free energy relationship analysis. The results suggest that the coarse-grained AChR gating conformational change propagates through the protein with dynamics that are governed by the Brownian motion of individual gating blocks.
Synchronizing to real events: Subjective audiovisual alignment scales with perceived auditory depth and speed of sound
Authors: Alais David , Carlile Simon
Published: January 2005

Because of the slow speed of sound relative to light, acoustic and visual signals from a distant event often will be received asynchronously. Here, using acoustic signals with a robust cue to sound source distance, we show that judgments of perceived temporal alignment with a visual marker depend on the depth simulated in the acoustic signal. For distant sounds, a large delay of sound relative to vision is required for the signals to be perceived as temporally aligned. For nearer sources, the time lag corresponding to audiovisual alignment is smaller and scales at rate approximating the speed of sound. Thus, when robust cues to auditory distance are present, the brain can synchronize disparate audiovisual signals to external events despite considerable differences in time of arrival at the perceiver. This ability is functionally important as it allows auditory and visual signals to be synchronized to the external event that caused them.
Phosphodiesterase-5A dysregulation in penile erectile tissue is a mechanism of priapism
Authors: Champion Hunter C. , Bivalacqua Trinity J. , Takimoto Eiki , Kass David A. , Burnett Arthur L.
Published: January 2005

The molecular mechanism for priapism is not well characterized. Although the nitric oxide (NO) pathway is known to mediate penile erection under normal conditions, we hypothesized that the mechanism of priapism rests in aberrant downstream signaling of this pathway based on our previous findings that mice lacking the gene for endothelial nitric oxide synthase (eNOS—/—) and mice lacking both neuronal NOS (nNOS) and eNOS (nNOS—/—, eNOS—/—) have a tendency for priapic activity. We investigated the role of downstream guanylate cyclase and phosphodiesterase type 5 (PDE5A) expression and function in mediating these responses in eNOS—/—and nNOS—/—, eNOS—/—mice. Erectile responses to both cavernous nerve stimulation and intracavernosal injection of the NO donor diethylamine-NONOate were augmented in eNOS—/—and nNOS—/—, eNOS—/—mice but not in WT or nNOS—/—mice. PDE5A protein expression and activity and cGMP levels were significantly lower in eNOS—/—and nNOS—/—, eNOS—/—mice, and this effect was reproduced in WT corpus cavernosum exposed to NOS inhibitors. Moreover, cavernous nerve stimulation was associated with a marked augmentation of cavernosal cGMP levels, suggesting that, although lower at baseline, the production of cGMP is unchecked in eNOS—/—and nNOS—/—, eNOS—/—mice upon neurostimulation. Transfection of eNOS—/—mice with an adenovirus encoding eNOS resulted in a normalization of PDE5A protein and activity as well as a correction of priapic activity. Coupled with the observation that sickle cell disease mice (which show a priapism phenotype) evince dysregulated PDE5A expression/activity, these data suggest that PDE5A dysregulation is a fundamental mechanism for priapism.
Microengineered hydromechanical cochlear model
Authors: White Robert D. , Grosh Karl
Published: January 2005

Micromachined fluid-filled variable impedance waveguides intended to mimic the mechanics of the passive mammalian cochlea have been fabricated and experimentally examined. The structures were microfabricated with dimensions similar to those of the biological system. Experimental tests demonstrate acoustically excited traveling fluid-structure waves with phase accumulations between 1.5 and 3 π radians at the location of maximum response. The resulting measured frequency-position mapping function, with similarities to that observed in the cochlea, is presented. Results for both isotropic and orthotropic membranes are reported, demonstrating that the achieved orthotropy ratio of 8:1 in tension is insufficient to produce the sharp filtering observed in animal experiments and many computational models that use higher ratios. It is also shown experimentally that high viscosity fluids must be used to provide sufficient damping to avoid the formation of a nonphysiological standing wave pattern. A mathematical model incorporating a thin-layer viscous, compressible fluid approximation coupled to an orthotropic membrane model is validated against experimental results. The work presented herein is motivated by the possibility of producing microfabricated cochlear-like filters, thus the structure is designed for production in a scalable microfabrication process.
Transient interference with staphylococcal quorum sensing blocks abscess formation
Authors: Wright Jesse S. , Jin Rhuzong , Novick Richard P.
Published: January 2005

The staphylococcal virulon is controlled largely by theagrlocus, a global accessory gene regulator that is autoinduced by a self-coded peptide (AIP) and is therefore a quorum sensor. Theagrlocus has diverged within and between species, giving rise to AIP variants that inhibit heterologousagractivation, an effect with therapeutic potential againstStaphylococcus aureus: a single dose of an inhibitory AIP blocks the formation of an experimental murine abscess. As the AIP is unstable at physiological pH, owing to its essential thiolactone bond, its single-dose efficacy seems paradoxical, which has led us to analyze thein vivokinetics ofagractivation and the consequences of its blockage by a heterologous AIP. Initially, the infecting bacteria grow rapidly, achieving sufficient population density within the first 3 h to activateagr, and then enter a neutrophil-induced metabolic eclipse lasting for 2–3 d, followed byagrreactivation concomitantly with the development of the abscess. The inhibitory AIP preventsagrexpression only during its shortin vivolifetime, suggesting that theagr-induced and therefore quorum-dependent synthesis of virulence factors shortly after infection is necessary for the subsequent development of the abscess lesion and bacterial survival. We confirm this finding by showing that a sterileagr+supernatant causes a sterile abscess similar to the septic abscess caused by live bacteria. These results may provide a biological rationale for regulation of virulence factor expression by quorum sensing rather than by response to specific host signals.
ATP-driven stepwise rotation of F
o
F
1
-ATP synthase
Authors: Ueno Hiroshi , Suzuki Toshiharu , Kinosita Kazuhiko , Yoshida Masasuke
Published: January 2005

F
o
F
1
-ATP synthase (F
o
F
1
) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F
1
, a water-soluble ATPase portion of F
o
F
1
, rotates by repeating ATP-waiting dwell, 80° substep rotation, catalytic dwell, and 40°-substep rotation. Compared with F
1
, rotation of F
o
F
1
has yet been poorly understood, and, here, we analyzed ATP-driven rotations of F
o
F
1
. Rotation was probed with an 80-nm bead attached to the ring of
c
subunits in the immobilized F
o
F
1
and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a
K
m
of ≈30 μM and a
V
max
of ≈350 revolutions per second (at 37°C). At low ATP, ATP-waiting dwell was seen and the
k
on-ATP
was estimated to be 3.6 × 10
7
M
-1
·s
-1
. At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5′-[γ-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of ≈80°. The rotational behavior of F
o
F
1
resembles that of F
1
. This finding indicates that “friction” in F
o
motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.
Fast decay of iconic memory in observers with mild cognitive impairments
Authors: Lu Zhong-Lin , Neuse James , Madigan Stephen , Dosher Barbara Anne
Published: January 2005

In a previous clinical report, unusually fast decay of iconic memory was obtained from a subject who later developed Alzheimer's disease. By using the partial-report paradigm, iconic memory (a form of visual sensory memory) in a group of observers with mild cognitive impairments (MCI) was characterized and compared with that of young college-age adults and older controls. Relatively long stimulus exposures were used for all three groups to ensure that older observers could perceive the stimuli. A set of conventional neuropsychological tests assessed cognitive functions of the MCI and older control groups. We found that iconic memory decayed much faster for observers with MCI than for normal controls, old or young, although the two groups of older observers performed at equivalent levels in precue tests (assay of visibility) and tests cued at long delays (assay of short-term memory). The result suggests that fast decay of iconic memory might be a general characteristic of observers with MCI who are at much higher than average risk of developing Alzheimer's disease later in life.
Accessing key steps of human tumor progression
in vivo
by using an avian embryo model
Authors: Hagedorn Martin , Javerzat Sophie , Gilges Delphine , Meyre Aurélie , de Lafarge Benjamin , Eichmann Anne , Bikfalvi Andreas
Published: January 2005

Experimental
in vivo
tumor models are essential for comprehending the dynamic process of human cancer progression, identifying therapeutic targets, and evaluating antitumor drugs. However, current rodent models are limited by high costs, long experimental duration, variability, restricted accessibility to the tumor, and major ethical concerns. To avoid these shortcomings, we investigated whether tumor growth on the chick chorio-allantoic membrane after human glioblastoma cell grafting would replicate characteristics of the human disease. Avascular tumors consistently formed within 2 days, then progressed through vascular endothelial growth factor receptor 2-dependent angiogenesis, associated with hemorrhage, necrosis, and peritumoral edema. Blocking of vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor signaling pathways by using small-molecule receptor tyrosine kinase inhibitors abrogated tumor development. Gene regulation during the angiogenic switch was analyzed by oligonucleotide microarrays. Defined sample selection for gene profiling permitted identification of regulated genes whose functions are associated mainly with tumor vascularization and growth. Furthermore, expression of known tumor progression genes identified in the screen (IL-6 and cysteine-rich angiogenic inducer 61) as well as potential regulators (lumican and F-box-only 6) follow similar patterns in patient glioma. The model reliably simulates key features of human glioma growth in a few days and thus could considerably increase the speed and efficacy of research on human tumor progression and preclinical drug screening.
Regular multicharged transient soft matter in Coulomb explosion of heteroclusters
Authors: Last Isidore , Jortner Joshua
Published: January 2005

Nanointerfaces of mobile, thin spherical shells of light ions that expand on the femtosecond time scale, can be produced by Coulomb explosion of extremely ionized molecular heteroclusters consisting of light and heavy ions, e.g., (D
+
I

q

+
)

n

(
q
= 7-35), which are generated in ultraintense laser fields (intensity,
I
, = 10
16
to 10
20
W·cm
-2
). Modeling, together with molecular dynamics simulations, reveals the expansion of 2D monolayers with high energies and narrow energy distributions [e.g.,
E
av
≃ 23 keV and Δ
E/E
av
= 0.16 for D
+
from (D
+
I
25+
)
2171
] arising from kinematic run-over effects. The expanding regular, monoionic, spherical nanointerfaces manifest the attainment of transient self-organization in complex systems driven by repulsive Coulomb interactions.
Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells
Authors: Wu Douglass , Xing Guo-Wen , Poles Michael A. , Horowitz Amir , Kinjo Yuki , Sullivan Barbara , Bodmer-Narkevitch Vera , Plettenburg Oliver , Kronenberg Mitchell , Tsuji Moriya , Ho David D. , Wong Chi-Huey
Published: January 2005

The CD1 family of proteins binds self and foreign glycolipids for presentation to CD1-restricted T cells. To identify previously uncharacterized active CD1 ligands, especially those of microbial origin, numerous glycolipids were synthesized and tested for their ability to stimulate mouse and human natural killer T (NKT) cells. They included analogs of the well known NKT cell agonist α-galactosyl ceramide (α-GalCer), bacterial glycolipids, and variations of the self-glycolipid, sulfatide. Bacterial glycolipids, α-galacturonosyl-ceramides fromSphingomonas wittichii, although structurally similar to α-GalCer, have significant differences in the sugar head group as well as the ceramide portion. TheSphingomonasglycosphingolipids (GSLs) and sulfatide variants were shown to activate human NKT cells as measured by IL-4 and IFN-γ secretion. Moreover, CD1d-dimer staining revealed human NKT cell reactivity toward these GSLs and to the sulfatides in a fashion comparable with α-GalCer. Because α-GalCer is a marine-sponge-derived ligand, our study here shows that bacterium-derived antigens are also able to stimulate mouse and human NKT cells.
Feedbacks and the coevolution of plants and atmospheric CO
2
Authors: Beerling David J. , Berner Robert A.
Published: January 2005

The coupled evolution of land plants, CO
2
, and climate over the last half billion years has maintained atmospheric CO
2
concentrations within finite limits, indicating the involvement of a complex network of geophysiological feedbacks. But insight into this important regulatory network is extremely limited. Here we present a systems analysis of the physiological and geochemical processes involved, identifying new positive and negative feedbacks between plants and CO
2
on geological time scales. Positive feedbacks accelerated falling CO
2
concentrations during the evolution and diversification of terrestrial ecosystems in the Paleozoic and enhanced rising CO
2
concentrations across the Triassic–Jurassic boundary during flood basalt eruptions. The existence of positive feedbacks reveals the unexpected destabilizing influence of the biota in climate regulation that led to environmental modifications accelerating rates of terrestrial plant and animal evolution in the Paleozoic.
Experiments investigating cooperative types in humans: A complement to evolutionary theory and simulations
Authors: Kurzban Robert , Houser Daniel
Published: January 2005

Unlike other species, humans cooperate in large, distantly related groups, a fact that has long presented a puzzle to biologists. The pathway by which adaptations for large-scale cooperation among nonkin evolved in humans remains a subject of vigorous debate. Results from theoretical analyses and agent-based simulations suggest that evolutionary dynamics need not yield homogeneous populations, but can instead generate a polymorphic population that consists of individuals who vary in their degree of cooperativeness. These results resonate with the recent increasing emphasis on the importance of individual differences in understanding and modeling behavior and dynamics in experimental games and decision problems. Here, we report the results of laboratory experiments that complement both theory and simulation results. We find that our subjects fall into three types, an individual's type is stable, and a group's cooperative outcomes can be remarkably well predicted if one knows its type composition. Reciprocal types, who contribute to the public good as a positive function of their beliefs about others' contributions, constitute the majority (63%) of players; cooperators and free-riders are also present in our subject population. Despite substantial behavioral differences, earnings among types are statistically identical. Our results support the view that our human subject population is in a stable, polymorphic equilibrium of types.
Differential contribution of the three Aph1 genes to γ-secretase activity
in vivo
Authors: Serneels Lutgarde , Dejaegere Tim , Craessaerts Katleen , Horré Katrien , Jorissen Ellen , Tousseyn Thomas , Hébert Sébastien , Coolen Marcel , Martens Gerard , Zwijsen An , Annaert Wim , Hartmann Dieter , De Strooper Bart
Published: January 2005

γ-Secretase is the protease responsible for amyloid β peptide release and is needed for Notch, N-Cadherin, and possibly other signaling pathways. The protease complex consists of at least four subunits, i.e., Presenilin, Aph1, Pen2, and Nicastrin. Two different genes encode Aph1A and Aph1B in man. A duplication of
Aph1B
in rodents has given rise to a third gene,
Aph1C
. Different mixes of γ-secretase subunits assemble in at least four human and six rodent complexes but it is not known whether they have different activities
in vivo
. We report here the inactivation of the three
Aph1
genes in mice.
Aph1A
–/–
embryos show a lethal phenotype characterized by angiogenesis defects in the yolk sac, neuronal tube malformations, and mild somitogenesis defects.
Aph1B
–/–
or
C
–/–
or the combined
Aph1BC
–/–
mice (which can be considered as a model for total
Aph1B
loss in human) survive into adulthood. However,
Aph1BC
–/–
deficiency causes a mild but significant reduction in amyloid β percursor protein processing in selective regions of the adult brain. We conclude that the biochemical and physiological repercussions of genetically reducing γ-secretase activity via the different
Aph1
components are quite divergent and tissue specific. Our work provides
in vivo
evidence for the concept that different γ-secretase complexes may exert different biological functions. In the context of Alzheimer's disease therapy, this implies the theoretical possibility that targeting specific γ-secretase subunit combinations could yield less toxic drugs than the currently available general inhibitors of γ-secretase activity.
T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses
Authors: Buono Chiara , Binder Christoph J. , Stavrakis George , Witztum Joseph L. , Glimcher Laurie H. , Lichtman Andrew H.
Published: January 2005

The influence of the immune system on atherosclerosis involves both helper T (Th) cell and antibody responses to plaque antigens. These responses may have proatherogenic and protective effects. T-bet is a transcription factor required for Th1 differentiation and regulates the balance between Th1 and Th2 responses in inflammatory diseases. To clarify how helper T cell subset differentiation influences atherosclerosis, we compared lesion development and immune responses to plaque antigens in low-density lipoprotein receptor-deficient (
Ldlr
-/-
) mice with or without functional
T-bet
genes. Atherosclerosis was significantly reduced in T-bet-deficient
Ldlr
-/-
mice compared with
Ldlr
-/-
controls, and the lesions that did develop in the absence of T-bet had less smooth muscle cell content. Furthermore, T-bet deficiency caused a Th2 switch in the response to the atherosclerosis-associated antigen heat shock protein-60, and a change in T-dependent isotypes of oxidized LDL-specific antibodies. Of particular significance, T-bet deficiency caused a >250% increase in the titer of E06 antibodies, which are known to be atheroprotective and whose production by B-1 B cells is enhanced by IL-5. These findings establish that T cell subset differentiation influences both T cell and antibody responses that modulate atherosclerosis, and validate the therapeutic goal of skewing T responses to atherosclerosis-associated antigens.
Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in
Synechococcus
and
Arabidopsis
Authors: Waditee Rungaroon , Bhuiyan Md. Nazmul H. , Rai Vandna , Aoki Kenji , Tanaka Yoshito , Hibino Takashi , Suzuki Shigetoshi , Takano Jun , Jagendorf André T. , Takabe Tetsuko , Takabe Teruhiro
Published: January 2005

Betaine is an important osmoprotectant, synthesized by many plants in response to abiotic stresses. Almost all known biosynthetic pathways of betaine are two-step oxidations of choline. Recently, a biosynthetic pathway of betaine from glycine, catalyzed by two
N
-methyltransferase enzymes, was found. Here, the potential role of
N
-methyltransferase genes for betaine synthesis was examined in a freshwater cyanobacterium,
Synechococcus
sp. PCC 7942, and in
Arabidopsis
plants. It was found that the coexpression of
N
-methyltransferase genes in
Synechococcus
caused accumulation of a significant amount of betaine and conferred salt tolerance to a freshwater cyanobacterium sufficient for it to become capable of growth in seawater.
Arabidopsis
plants expressing
N
-methyltransferase genes also accumulated betaine to a high level in roots, stems, leaves, and flowers and improved seed yield under stress conditions. Betaine levels were higher than those produced by choline-oxidizing enzymes. These results demonstrate the usefulness of glycine
N
-methyltransferase genes for the improvement of abiotic stress tolerance in crop plants.
Peptide 15-mers of defined sequence that substitute for random amino acid copolymers in amelioration of experimental autoimmune encephalomyelitis
Authors: Stern Joel N. H. , Illés Zsolt , Reddy Jayagopala , Keskin Derin B. , Fridkis-Hareli Masha , Kuchroo Vijay K. , Strominger Jack L.
Published: January 2005

Myelin basic protein (MBP) is a major candidate autoantigen in multiple sclerosis (MS). Its immunodominant epitope, MBP 85–99, forms a complex with human leukocyte antigen (HLA)-DR2 with which multiple sclerosis is genetically associated. Copolymer 1 (Copaxone), a random amino acid copolymer [poly (Y,E,A,K)n] as well as two modified synthetic copolymers [poly (F,Y,A,K)n and poly (V,W,A,K)n] also form complexes with HLA-DR2 (DRA/DRB1*1501) and compete with MBP 85–99 for binding. Moreover, two high-affinity synthetic peptide 15-mers that could inhibit binding even more effectively were previously designed. Here, we show that further-modified peptide 15-mers inhibited even more strongly (in order J5 > J3 > J2) both the binding of MBP 85–99 to HLA-DR2 and IL-2 production by two MBP 85–99-specific HLA-DR2-restricted T cells. J5, J3, and J2 also suppressed both MBP 85–99-induced experimental autoimmune encephalomyelitis (EAE) in humanized mice and proteolipid protein 139–151-induced EAE in SJL/J mice. Moreover, none of these previously uncharacterized peptide inhibitors crossreacted with MBP 85–99- or proteolipid protein 139–151-specific T cells. In both cases, spleen and lymph node cultures stimulated with these peptides produced large amounts of Th2 cytokines (IL-4 and IL-10), and adoptive transfer of established T cell lines suppressed disease induction. These peptide 15-mers provide specific, nonrandom sequences that appear to be at least as effective as random copolymers in suppressing EAE in several models.
Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin αXβ2
Authors: Vorup-Jensen Thomas , Carman Christopher V. , Shimaoka Motomu , Schuck Peter , Svitel Juraj , Springer Timothy A.
Published: January 2005

The structural integrity of tissue proteins is damaged in processes ranging from remodeling of the extracellular matrix to destruction by microbial pathogens. Leukocytes play a prominent role in tissue surveillance and repair. However, it remains enigmatic what features of structurally decayed proteins prompt recognition by leukocyte cell-surface receptors. Here, we report that adhesion of human neutrophil granulocytes to fibrinogen is greatly increased by plasmin digestion in a mode where αXβ2dominates the integrin-dependent binding. The bacterial protease subtilisin also enhances binding by αXβ2. The αXligand binding domain has an unusually high affinity for carboxyl groups, withKDat ≈100 μM. Our findings implicate enhanced accessibility of negatively charged residues in structurally decayed proteins as a pattern recognition motif for αXβ2integrin. Comparisons among integrins show relevance of these findings to the large number of ligands recognized by αMβ2and αXβ2but not αLβ2. The observations suggest that the pericellular proteolysis at the leading edge of neutrophils not only facilitates passage through the extracellular matrix but also manufactures binding sites for αXβ2.
Unparalleled complexity of the MHC class I region in rhesus macaques
Authors: Otting Nel , Heijmans Corrine M. C. , Noort Riet C. , de Groot Natasja G. , Doxiadis Gaby G. M. , van Rood Jon J. , Watkins David I. , Bontrop Ronald E.
Published: January 2005

The highly polymorphic gene products of the classical MHC class I genes in humans (HLA-A,HLA-B,andHLA-C) play a critical role in the immune defense against intracellular infections. Because non-human primates are important models for AIDS vaccine research, rhesus monkeys from a thoroughly pedigreed and serotyped colony were subjected to full-length cDNA analysis of MHC class I gene transcripts. Rhesus macaques express multiple dominantMamu-AandMamu-Btranscripts (majors) per chromosome, which are characterized by high expression levels. The presence of additional cDNAs with low levels of expression (minors) suggests evidence for transcriptional control of MHC class I genes. Moreover, phylogenetic analyses illustrate that most of theMamu-AandMamu-Bloci/lineages identified display no or only limited levels of allelic polymorphism. Thus, MHC class I diversity in rhesus macaques is typified by the existence of an unmatched high number ofMamu-A and Mamu-Bregion configurations that exhibit polymorphism with regard to the number and combination of transcribed loci present per chromosome.
Impaired intranuclear trafficking of Runx2 (AML3/CBFA1) transcription factors in breast cancer cells inhibits osteolysis
in vivo
Authors: Javed Amjad , Barnes George L. , Pratap Jitesh , Antkowiak Tomasz , Gerstenfeld Louis C. , van Wijnen Andre J. , Stein Janet L. , Lian Jane B. , Stein Gary S.
Published: January 2005

Runx transcription factors comprise a family of proteins that are essential for organogenesis. A unique nuclear matrix-targeting signal in the C terminus directs these factors to their appropriate subnuclear domains. At these sites, they interact with coregulatory proteins and target genes. We have previously shown that aberrant expression of the Runx2 DNA binding domain in metastatic breast cancer cells can prevent production of osteolytic lesions in bone. Here, we show that proper Runx2 subnuclear targeting is required for osteolysis. We have identified point mutations of the Runx2 nuclear matrix-targeting signal sequence that impair its targeting to nuclear matrix sites. These mutations block the invasive and osteolytic properties of MDA-MB-231 breast cancer cells
in vivo
. Cell lines expressing this Runx2 mutant protein inhibit the osteogenic properties of bone marrow stromal cells in coculture assays. The mutant breast cancer cells also exhibit reduced invasiveness
in vitro
and do not express genes involved in invasion and angiogenesis (VEGF and MMP13). Our findings suggest that fidelity of Runx2 intranuclear organization is necessary for expression of target genes that mediate the osteolytic activity of metastatic breast cancer cells.
Fast and reliable prediction of noncoding RNAs
Authors: Washietl Stefan , Hofacker Ivo L. , Stadler Peter F.
Published: January 2005

We report an efficient method for detecting functional RNAs. The approach, which combines comparative sequence analysis and structure prediction, already has yielded excellent results for a small number of aligned sequences and is suitable for large-scale genomic screens. It consists of two basic components: (
i
) a measure for RNA secondary structure conservation based on computing a consensus secondary structure, and (
ii
) a measure for thermodynamic stability, which, in the spirit of a
z
score, is normalized with respect to both sequence length and base composition but can be calculated without sampling from shuffled sequences. Functional RNA secondary structures can be identified in multiple sequence alignments with high sensitivity and high specificity. We demonstrate that this approach is not only much more accurate than previous methods but also significantly faster. The method is implemented in the program
rnaz
, which can be downloaded from
www.tbi.univie.ac.at/~wash/RNAz
. We screened all alignments of length
n
≥ 50 in the Comparative Regulatory Genomics database, which compiles conserved noncoding elements in upstream regions of orthologous genes from human, mouse, rat,
Fugu
, and zebrafish. We recovered all of the known noncoding RNAs and cis-acting elements with high significance and found compelling evidence for many other conserved RNA secondary structures not described so far to our knowledge.
A cascade of cytokines mediates mechanical inflammatory hypernociception in mice
Authors: Cunha T. M. , Verri W. A. , Silva J. S. , Poole S. , Cunha F. Q. , Ferreira S. H.
Published: January 2005

The hypernociceptive effects of cytokines [TNF-α, keratinocyte-derived chemokine (KC), and IL-1β] and their participation in carrageenan (Cg)-induced inflammatory hypernociception in mice were investigated. Nociceptor sensitization (hypernociception) was quantified with an electronic version of the von Frey filament test in WT and TNF receptor type 1 knockout mice (TNF-R1
–/–
). TNF-α-induced hypernociception was abolished in TNF-R1
–/–
mice, partially inhibited by pretreatment with IL-1 receptor antagonist (IL-1ra) or indomethacin and unaffected by Ab against KC (AbKC) or guanethidine. IL-1ra and indomethacin pretreatment strongly inhibited the hypernociception induced by IL-1β, which was not altered by AbKC or guanethidine or by knocking out TNF-R1. KC-induced hypernociception was abolished by AbKC, inhibited by pretreatment with indomethacin plus guanethidine, and partially inhibited by IL-1ra, indomethacin, or guanethidine. In contrast, KC-induced hypernociception was not altered by knocking out TNF-R1. Cg-induced hypernociception was abolished by administration of indomethacin plus guanethidine, diminished in TNF-R1
–/–
mice, and partially inhibited in WT mice pretreated with AbKC, IL-1ra, indomethacin, or guanethidine. TNF-α, KC, and IL-1β concentrations were elevated in the skin of Cg-injected paws. The TNF-α and KC concentrations rose concomitantly and peaked before that of IL-1β. In mice, the cytokine cascade begins with the release of TNF-α (acting on TNF-R1 receptor) and KC, which stimulate the release of IL-1β. As in rats, the final mediators of this cascade were prostaglandins released by IL-1β and sympathetic amines released by KC. These results extend to mice the concept that the release of primary mediators responsible for hypernociception is preceded by a cascade of cytokines.
Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity
Authors: Forouhar Farhad , Yang Yue , Kumar Dhirendra , Chen Yang , Fridman Eyal , Park Sang Wook , Chiang Yiwen , Acton Thomas B. , Montelione Gaetano T. , Pichersky Eran , Klessig Daniel F. , Tong Liang
Published: January 2005

Salicylic acid (SA) is a critical signal for the activation of plant defense responses against pathogen infections. We recently identified SA-binding protein 2 (SABP2) from tobacco as a protein that displays high affinity for SA and plays a crucial role in the activation of systemic acquired resistance to plant pathogens. Here we report the crystal structures of SABP2, alone and in complex with SA at up to 2.1-Å resolution. The structures confirm that SABP2 is a member of the α/β hydrolase superfamily of enzymes, with Ser-81, His-238, and Asp-210 as the catalytic triad. SA is bound in the active site and is completely shielded from the solvent, consistent with the high affinity of this compound for SABP2. Our biochemical studies reveal that SABP2 has strong esterase activity with methyl salicylate as the substrate, and that SA is a potent product inhibitor of this catalysis. Modeling of SABP2 with MeSA in the active site is consistent with all these biochemical observations. Our results suggest that SABP2 may be required to convert MeSA to SA as part of the signal transduction pathways that activate systemic acquired resistance and perhaps local defense responses as well.
Metabolic engineering of dhurrin in transgenic
Arabidopsis
plants with marginal inadvertent effects on the metabolome and transcriptome
Authors: Kristensen Charlotte , Morant Marc , Olsen Carl Erik , Ekstrøm Claus T. , Galbraith David W. , Lindberg Møller Birger , Bak Søren
Published: January 2005

Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in
Arabidopsis thaliana
by genetic engineering. Transgenic
A. thaliana
plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of
CYP79A1
,
CYP71E1
, and
UGT85B1
from
Sorghum bicolor
were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only
CYP79A1
accumulated 3% dry weight of the tyrosine-derived glucosinolate,
p
-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of
CYP79A1
plus
CYP71E1
resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with
S. bicolor UGT85B
. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic
A. thaliana
plants with marginal inadvertent effects on the transcriptome and metabolome.
Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases
Authors: Saiardi Adolfo , Resnick Adam C. , Snowman Adele M. , Wendland Beverly , Snyder Solomon H.
Published: January 2005

Inositol pyrophosphates physiologically regulate vesicular endocytosis, ribosomal disposition, and directly phosphorylate proteins. Here we demonstrate roles in cell death and regulation of telomere length. Lethal actions of wortmannin and caffeine are selectively abolished in yeast mutants that cannot synthesize inositol pyrophosphates. Wortmannin and caffeine appear to act through the phosphoinositide 3-kinase-related protein kinases Tel1 and Mec1, known regulators of telomere length. Inositol pyrophosphates physiologically antagonize the actions of these kinases, which is demonstrated by the fact that yeast mutants with reduced or elevated levels of inositol pyrophosphates, respectively, display longer and shorter telomeres.
Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54
Authors: Messager Sophie , Chatzidaki Emmanouella E. , Ma Dan , Hendrick Alan G. , Zahn Dirk , Dixon John , Thresher Rosemary R. , Malinge Isabelle , Lomet Didier , Carlton Mark B. L. , Colledge William H. , Caraty Alain , Aparicio Samuel A. J. R.
Published: January 2005

We have recently described a molecular gatekeeper of the hypothalamic-pituitary-gonadal axis with the observation that G protein-coupled receptor 54 (GPR54) is required in mice and men for the pubertal onset of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion to occur. In the present study, we investigate the possible central mode of action of GPR54 and kisspeptin ligand. First, we show that GPR54 transcripts are colocalized with gonadotropin-releasing hormone (GnRH) neurons in the mouse hypothalamus, suggesting that kisspeptin, the GPR54 ligand, may act directly on these neurons. Next, we show that GnRH neurons seem anatomically normal in gpr54
–/–
mice, and that they show projections to the median eminence, which demonstrates that the hypogonadism in gpr54
–/–
mice is not due to an abnormal migration of GnRH neurons (as occurs with KAL1 mutations), but that it is more likely due to a lack of GnRH release or absence of GnRH neuron stimulation. We also show that levels of kisspeptin injected i.p., which stimulate robust LH and FSH release in wild-type mice, have no effect in gpr54
–/–
mice, and therefore that kisspeptin acts directly and uniquely by means of GPR54 signaling for this function. Finally, we demonstrate by direct measurement, that the central administration of kisspeptin intracerebroventricularly in sheep produces a dramatic release of GnRH into the cerebrospinal fluid, with a parallel rise in serum LH, demonstrating that a key action of kisspeptin on the hypothalamo-pituitary-gonadal axis occurs directly at the level of GnRH release. The localization and GnRH release effects of kisspeptin thus define GPR54 as a major control point in the reproductive axis and suggest kisspeptin to be a neurohormonal effector.
Crystal structure of a pH-regulated luciferase catalyzing the bioluminescent oxidation of an open tetrapyrrole
Authors: Schultz L. Wayne , Liu Liyun , Cegielski Margaret , Hastings J. Woodland
Published: January 2005

The luciferase ofLingulodinium polyedrum, a marine bioluminescent dinoflagellate, consists of three similar but not identical domains in a single polypeptide. Each encodes an active luciferase that catalyzes the oxidation of a chlorophyll-derived open tetrapyrrole (dinoflagellate luciferin) to produce blue light. These domains share no sequence similarity with any other in the GenBank database and no structural or motif similarity with any other luciferase. We report here the 1.8-Å crystal structure of the third domain, D3, at pH 8, and a mechanism for its activity regulation by pH. D3 consists of two major structural elements: a β-barrel pocket putatively for substrate binding and catalysis and a regulatory three-helix bundle. N-terminal histidine residues previously shown to regulate activity by pH are at the interface of the helices in the bundle. Molecular dynamics calculations indicate that, in response to changes in pH, these histidines could trigger a large molecular motion of the bundle, thereby exposing the active site to the substrate.
Enhancer-binding proteins with a forkhead-associated domain and the σ
54
regulon in
Myxococcus xanthus
fruiting body development
Authors: Jelsbak Lars , Givskov Michael , Kaiser Dale
Published: January 2005

In response to starvation,
Myxococcus xanthus
initiates a developmental program that results in the formation of spore-filled, multicellular fruiting bodies. Many developmentally regulated genes in
M. xanthus
are transcribed from σ
54
promoters, and these genes require enhancer-binding proteins. Here we report the finding of an unusual group of 12 genes encoding σ
54
-dependent enhancer-binding proteins containing a forkhead-associated (FHA) domain as their N-terminal sensory domain. FHA domains in other proteins recognize phosphothreonine residues. An insertion mutation in one of these genes,
Mx4885
, caused a cell autonomous aggregation and sporulation defect. In-frame deletion mutants showed that the FHA domain is necessary for proper Mx4885 function. The altered pattern of developmental gene expression in the mutant implied that Mx4885 is on the pathway of response to the morphogenetic C-signal. Immunoblots specific for C-signal and FruA imply that the site of Mx4885 action is downstream of FruA synthesis on the C-signal transduction pathway. Mx4885 may help to coordinate the level of intracellular phosphorylated FruA (FruA-P) with the level of C-signal displayed on the signal donor cell. Because FHA domains respond to phosphothreonine-containing proteins, these results suggest a regulatory link to the abundant Ser/Thr protein kinases in
M. xanthus
.
Iron release from macrophages after erythrophagocytosis is up-regulated by ferroportin 1 overexpression and down-regulated by hepcidin
Authors: Knutson Mitchell D. , Oukka Mohamed , Koss Lindsey M. , Aydemir Fikret , Wessling-Resnick Marianne
Published: January 2005

Ferroportin 1 (FPN1) is transmembrane protein involved in iron homeostasis. In the duodenum, FPN1 localizes to the basolateral surface of enterocytes where it appears to export iron out of the cell and into the portal circulation. FPN1 is also abundantly expressed in reticuloendothelial macrophages of the liver, spleen, and bone marrow, suggesting that this protein serves as an iron exporter in cells that recycle iron from senescent red blood cells. To directly test the hypothesis that FPN1 functions in the export of iron after erythrophagocytosis, FPN1 was stably expressed in J774 mouse macrophages by using retroviral transduction, and release of59Fe after phagocytosis of59Fe-labeled rat erythrocytes was measured. J774 cells overexpressing FPN1 released 70% more59Fe after erythrophagocytosis than control cells, consistent with a role in the recycling of iron from senescent red cells. Treatment of cells with the peptide hormone hepcidin, a systemic regulator of iron metabolism, dramatically decreased FPN1 protein levels and significantly reduced the efflux of59Fe after erythrophagocytosis. Subsequent fractionation of the total released59Fe into heme and nonheme compounds revealed that hepcidin treatment reduced the release of nonheme59Fe by 50% and 25% from control and FPN1-overexpressing cells, respectively, but did not diminish efflux of59Fe-heme. We conclude that FPN1 is directly involved in the export of iron during erythrocyte-iron recycling by macrophages.