bluuebunny/crossref_metadata_2025 huggingface dataset:
Combinatorial interactions of Serpent, Lozenge, and U-shaped regulate crystal cell lineage commitment during
Drosophila
hematopoiesis
Authors: Fossett Nancy , Hyman Kristy , Gajewski Kathleen , Orkin Stuart H. , Schulz Robert A.
Published: October 2003

The GATA factor Serpent (Srp) is required for hemocyte precursor formation during
Drosophila
hematopoiesis. These blood cell progenitors give rise to two distinct lineages within the developing embryo. Lozenge, a Runx protein homologue, and Glial cells missing-1 and -2 are essential for crystal cell and plasmatocyte production, respectively. In contrast U-shaped, a Friend of GATA class factor, antagonizes crystal cell formation. Here we show that Srp, Lozenge, and U-shaped interact in different combinations to regulate crystal cell lineage commitment. Coexpression of Srp and Lozenge synergistically activated the crystal cell program in both embryonic and larval stages. Furthermore, expression of Lozenge and SrpNC, a Srp isoform with N- and C-terminal zinc fingers, inhibited
u-shaped
expression, indicating that crystal cell activation coincided with the down-regulation of this repressor-encoding gene. In contrast, whereas U-shaped and SrpNC together blocked crystal cell production, coexpression of U-shaped with noninteracting Srp proteins failed to prevent overproduction of this hemocyte population. Such results indicated that U-shaped and SrpNC must interact to block crystal cell production. Taken together, these studies show that the specialized SrpNC isoform plays a pivotal role during crystal cell lineage commitment, acting as an activator or repressor depending on the availability of specific transcriptional coregulators. These findings provide definitive proof of the combinatorial regulation of hematopoiesis in
Drosophila
and an
in vivo
demonstration of GATA and Runx functional interaction in a blood cell commitment program.
Role of residual structure in the unfolded state of a thermophilic protein
Authors: Robic Srebrenka , Guzman-Casado Mercedes , Sanchez-Ruiz Jose M. , Marqusee Susan
Published: October 2003

Ribonucleases H from the thermophilic bacteriumThermus thermophilusand the mesophileEscherichia colidemonstrate a dramatic and surprising difference in their change in heat capacity upon unfolding (ΔCp°). The lower ΔCp° of the thermophilic protein directly contributes to its higher thermal denaturation temperature (Tm). We propose that this ΔCp° difference originates from residual structure in the unfolded state of the thermophilic protein; we verify this hypothesis by using a mutagenic approach. Residual structure in the unfolded state may provide a mechanism for balancing a highTmwith the optimal thermodynamic stability for a protein's function. Structure in the unfolded state is shown to differentially affect the thermodynamic profiles of thermophilic and mesophilic proteins.
Antiangiogenic proteins require plasma fibronectin or vitronectin for
in vivo
activity
Authors: Yi Ming , Sakai Takao , Fässler Reinhard , Ruoslahti Erkki
Published: October 2003

Fragmentation of various extracellular matrix and blood proteins generates antiangiogenic substances that are physiological regulators of angiogenesis. Some of these compounds are in clinical trials as inhibitors of tumor angiogenesis. Anastellin, an antiangiogenic protein fragment derived from fibronectin, was unable to inhibit matrigel plug angiogenesis in mice that lack plasma fibronectin. Anastellin was fully active in mice that are null for vitronectin, which, like fibronectin, is a major adhesion protein in the blood. An antiangiogenic form of antithrombin showed the opposite pattern. The activity of endostatin was impaired in both fibronectin- and vitronectin-deficient mice. These results suggest a shared mechanism of action for antiangiogenic factors derived from extracellular matrix and plasma proteins: these factors form complexes with adhesion proteins in plasma to create an active antiangiogenic substance.
Global climate change and mammalian species diversity in U.S. national parks
Authors: Burns Catherine E. , Johnston Kevin M. , Schmitz Oswald J.
Published: October 2003

National parks and bioreserves are key conservation tools used to protect species and their habitats within the confines of fixed political boundaries. This inflexibility may be their “Achilles' heel” as conservation tools in the face of emerging global-scale environmental problems such as climate change. Global climate change, brought about by rising levels of greenhouse gases, threatens to alter the geographic distribution of many habitats and their component species. With these changes comes great uncertainty about the future ability of parks and protected areas to meet their conservation mandates. We report here on an analysis aimed at assessing the extent of mammalian species turnover that may be experienced in eight selected U.S. national parks if climate change causes mammalian species within the continental U.S. to relocate to new geographic locations. Due to species losses of up to 20% and drastic influxes of new species, national parks are not likely to meet their mandate of protecting current biodiversity within park boundaries. This approach represents a conservative prognosis. As species assemblages change, new interactions between species may lead to less predictable indirect effects of climate change, increasing the toll beyond that found in this study.
Exclusively targeting β-secretase to lipid rafts by GPI-anchor addition up-regulates β-site processing of the amyloid precursor protein
Authors: Cordy Joanna M. , Hussain Ishrut , Dingwall Colin , Hooper Nigel M. , Turner Anthony J.
Published: October 2003

β-Secretase (BACE, Asp-2) is a transmembrane aspartic proteinase responsible for cleaving the amyloid precursor protein (APP) to generate the soluble ectodomain sAPPβ and its C-terminal fragment CTFβ. CTFβ is subsequently cleaved by γ-secretase to produce the neurotoxic/synaptotoxic amyloid-β peptide (Aβ) that accumulates in Alzheimer's disease. Indirect evidence has suggested that amyloidogenic APP processing may preferentially occur in lipid rafts. Here, we show that relatively little wild-type BACE is found in rafts prepared from a human neuroblastoma cell line (SH-SY5Y) by using Triton X-100 as detergent. To investigate further the significance of lipid rafts in APP processing, a glycosylphosphatidylinositol (GPI) anchor has been added to BACE, replacing the transmembrane and C-terminal domains. The GPI anchor targets the enzyme exclusively to lipid raft domains. Expression of GPIBACE substantially up-regulates the secretion of both sAPPβ and amyloid-β peptide over levels observed from cells overexpressing wild-type BACE. This effect was reversed when the lipid rafts were disrupted by depleting cellular cholesterol levels. These results suggest that processing of APP to the amyloid-β peptide occurs predominantly in lipid rafts and that BACE is the rate-limiting enzyme in this process. The processing of the APP695isoform by GPI-BACE was up-regulated 20-fold compared with wild-type BACE, whereas only a 2-fold increase in the processing of APP751/770was seen, implying a differential compartmentation of the APP isoforms. Changes in the local membrane environment during aging may facilitate the cosegregation of APP and BACE leading to increased β-amyloid production.
A glucose sensor hiding in a family of transporters
Authors: Díez-Sampedro Ana , Hirayama Bruce A. , Osswald Christina , Gorboulev Valentin , Baumgarten Katharina , Volk Christopher , Wright Ernest M. , Koepsell Hermann
Published: October 2003

We have examined the expression and function of a previously undescribed human member (SGLT3/SLC5A4) of the sodium/glucose cotransporter gene family (SLC5) that was first identified by the chromosome 22 genome project. The cDNA was cloned and sequenced, confirming that the gene coded for a 659-residue protein with 70% amino acid identity to the human SGLT1. RT-PCR and Western blotting showed that the gene was transcribed and mRNA was translated in human skeletal muscle and small intestine. Immunofluorescence microscopy indicated that in the small intestine the protein was expressed in cholinergic neurons in the submucosal and myenteric plexuses, but not in enterocytes. In skeletal muscle SGLT3 immunoreactivity colocalized with the nicotinic acetylcholine receptor. Functional studies using the
Xenopus laevis
oocyte expression system showed that hSGLT3 was incapable of sugar transport, even though SGLT3 was efficiently inserted into the plasma membrane. Electrophysiological assays revealed that glucose caused a specific, phlorizin-sensitive, Na
+
-dependent depolarization of the membrane potential. Uptake assays under voltage clamp showed that the glucose-induced inward currents were not accompanied by glucose transport. We suggest that SGLT3 is not a Na
+
/glucose cotransporter but instead a glucose sensor in the plasma membrane of cholinergic neurons, skeletal muscle, and other tissues. This points to an unexpected role of glucose and SLC5 proteins in physiology, and highlights the importance of determining the tissue expression and function of new members of gene families.
Modification by docosahexaenoic acid of age-induced alterations in gene expression and molecular composition of rat brain phospholipids
Authors: Barceló-Coblijn Gwendolyn , Hőgyes Endre , Kitajka Klára , Puskás László G. , Zvara Ágnes , Hackler László , Nyakas Csaba , Penke Zsuzsa , Farkas Tibor
Published: October 2003

Advanced age is associated with reduced brain levels of long-chain polyunsaturated fatty acids, arachidonic acid (AA) and docosahexaenoic acid (DHA). Memory impairment is also a common phenomenon in this age. Two-year-old, essential fatty acid-sufficient rats were fed with fish oil (11% DHA) for 1 month, and fatty acid as well as molecular composition of the major phospholipids, phosphatidylcholine and phosphatidylethanolamine (PE), was compared with that of 2-month-old rats on the same diet. DHA but not AA was significantly reduced in brains of old rats but was restored to the level of young rats when they received rat chow fortified with fish oil. This effect was pronounced with diacyl 18:0/22:6 PE species, whereas levels of 18:1/22:6 and 16:0/22:6 remained unchanged in all of the three PE subclasses. Fish oil reduced the AA in the old rat brains, diacyl and alkenylacyl 18:0/20:4 PE being most affected. Phosphatidylcholines gave less pronounced response. Six genes were up-regulated, whereas no significant changes were observed in brains of old rats receiving fish oil for 1 month. None of them except synuclein in young rat brains could be related to mental functions. Old rats on the fish-oil diet did not perform better in Morris water maze test than the control ones. A 10% increase in levels of diacyl 18:0/22:6 PE in young rat brains resulted in a significant improvement of learning capacity. The results are interpreted in terms of the roles of different phospholipid molecular species in cognitive functions coupled with differential responsiveness of the genetic machinery of neurons to
n
-3 polyunsaturated fatty acids.
Maize-targeted mutagenesis: A knockout resource for maize
Authors: May Bruce P. , Liu Hong , Vollbrecht Erik , Senior Lynn , Rabinowicz Pablo D. , Roh Donna , Pan Xiaokang , Stein Lincoln , Freeling Mike , Alexander Danny , Martienssen Rob
Published: October 2003

We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F
1
plants were generated by using Robertson's
Mutator
(
Mu
) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10
–4
and 10
–5
per F
1
plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting
Mu
activity in most F
1
plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining
Mu
activity in the F
2
, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing
Mu
activity. These results have implications for epigenetic models of inbreeding and for functional genomics.
How protein thermodynamics and folding mechanisms are altered by the chaperonin cage: Molecular simulations
Authors: Takagi Fumiko , Koga Nobuyasu , Takada Shoji
Published: October 2003

How the
Escherichia coli
GroEL/ES chaperonin assists folding of a substrate protein remains to be uncovered. Recently, it was suggested that confinement into the chaperonin cage itself can significantly accelerate folding of a substrate. Performing comprehensive molecular simulations of eight proteins confined into various sizes
L
of chaperonin-like cage, we explore how and to what extent protein thermodynamics and folding mechanisms are altered by the cage. We show that a substrate protein is remarkably stabilized by confinement; the estimated increase in denaturation temperature Δ
T
f
is as large as ≈60°C. For a protein of size
R
0
, the stabilization Δ
T
f
scales as (
R
0
/
L
)
ν
, where ν ≈ 3, which is consistent with a mean field theory of polymer. We also found significant free energy cost of confining a protein, which increases with
R
0
/
L
, indicating that the confinement requires external work provided by the chaperonin system. In kinetic study, we show the folding is accelerated in a modestly well confined case, which is consistent with a recent experimental result on ribulose-1,5-bisphosphate carboxylase-oxygenase folding and simulation results of a β hairpin. Interestingly, the acceleration of folding is likely to be larger for a protein with more complex topology, as quantified by the contact order. We also show how ensemble of folding pathways are altered by the chaperonin-like cage calculating a variant of φ value used in the study of spontaneous folding.
Low monoamine oxidase B in peripheral organs in smokers
Authors: Fowler Joanna S. , Logan Jean , Wang Gene-Jack , Volkow Nora D. , Telang Frank , Zhu Wei , Franceschi Dinko , Pappas Naomi , Ferrieri Richard , Shea Colleen , Garza Victor , Xu Youwen , Schlyer David , Gatley S. John , Ding Yu-Shin , Alexoff David , Warner Donald , Netusil Noelwah , Carter Pauline , Jayne Millard , King Payton , Vaska Paul
Published: October 2003

One of the major mechanisms for terminating the actions of catecholamines and vasoactive dietary amines is oxidation by monoamine oxidase (MAO). Smokers have been shown to have reduced levels of brain MAO, leading to speculation that MAO inhibition by tobacco smoke may underlie some of the behavioral and epidemiological features of smoking. Because smoking exposes peripheral organs as well as the brain to MAO-inhibitory compounds, we questioned whether smokers would also have reduced MAO levels in peripheral organs. Here we compared MAO B in peripheral organs in nonsmokers and smokers by using positron emission tomography and serial scans with the MAO B-specific radiotracers,l-[11C]deprenyl and deuterium-substitutedl-[11C]deprenyl (l-[11C]deprenyl-D2). Binding specificity was assessed by using the deuterium isotope effect. We found that smokers have significantly reduced MAO B in peripheral organs, particularly in the heart, lungs, and kidneys, when compared with nonsmokers. Reductions ranged from 33% to 46%. Because MAO B breaks down catecholamines and other physiologically active amines, including those released by nicotine, its inhibition may alter sympathetic tone as well as central neurotransmitter activity, which could contribute to the medical consequences of smoking. In addition, although most of the emphases on the carcinogenic properties of smoke have been placed on the lungs and the upper airways, this finding highlights the fact that multiple organs in the body are also exposed to pharmacologically significant quantities of chemical compounds in tobacco smoke.
Molecular cloning and spatio-temporal expression of the prostaglandin transporter: A basis for the action of prostaglandins in the bovine reproductive system
Authors: Banu Sakhila K. , Arosh Joe A. , Chapdelaine Pierre , Fortier Michel A.
Published: October 2003

Prostaglandins (PGs) play important roles in mammalian reproductive function through autocrine, paracrine, and endocrine actions. However, they predominate as charged anions and diffuse poorly across the plasma membrane. Recently, a PG transporter (PGT) has been found to mediate PG transport across cell membranes. In ruminants, endometrial PGs are transported by a vascular pathway to the ovary to regress or rescue the corpus luteum. There is no report on the role of PGT in the reproductive functions of any species. We have cloned and characterized the bovine PGT (bPGT) that transports different PGs in the following affinity order: PGE
2
= PGF
2α
≥ PGD
2
much greater than arachidonate. bPGT mRNA and protein are expressed in endometrium, myometrium, and the utero-ovarian plexus (UOP) during the estrous cycle. The level of bPGT expression is higher in endometrium and UOP on the side of corpus luteum between days 13 and 18 of the estrous cycle. bPGT protein is localized in endometrial stroma, luminal epithelial cells, myometrial smooth muscle cells, and vascular smooth muscle cells of uterine vein and artery. In UOP, bPGT is selectively expressed in vascular smooth muscle cells of uterine vein and ovarian artery. Spatio-temporal expression of bPGT in uterine tissues and UOP supports a significant role of bPGT in cellular and compartmental transport of PGs to mediate the endocrine action at the time of luteolysis or establishment of pregnancy in bovine. This study describes and proposes a role of PGT in the regulation of reproductive processes.
Shock-wave cosmology inside a black hole
Authors: Smoller Joel , Temple Blake
Published: October 2003

We construct a class of global exact solutions of the Einstein equations that extend the Oppeheimer–Snyder model to the case of nonzero pressure, inside the black hole, by incorporating a shock wave at the leading edge of the expansion of the galaxies, arbitrarily far beyond the Hubble length in the Friedmann–Robertson–Walker (FRW) spacetime. Here the expanding FRW universe emerges be-hind a subluminous blast wave that explodes outward from the FRW center at the instant of the big bang. The total mass behind the shock decreases as the shock wave expands, and the entropy condition implies that the shock wave must weaken to the point where it settles down to an Oppenheimer–Snyder interface, (bounding a finite total mass), that eventually emerges from the white hole event horizon of an ambient Schwarzschild spacetime. The entropy condition breaks the time symmetry of the Einstein equations, selecting the explosion over the implosion. These shock-wave solutions indicate a cosmological model in which the big bang arises from a localized explosion occurring inside the black hole of an asymptotically flat Schwarzschild spacetime.
A candidate gene for developmental dyslexia encodes a nuclear tetratricopeptide repeat domain protein dynamically regulated in brain
Authors: Taipale Mikko , Kaminen Nina , Nopola-Hemmi Jaana , Haltia Tuomas , Myllyluoma Birgitta , Lyytinen Heikki , Muller Kurt , Kaaranen Minna , Lindsberg Perttu J. , Hannula-Jouppi Katariina , Kere Juha
Published: October 2003

Approximately 3–10% of people have specific difficulties in reading, despite adequate intelligence, education, and social environment. We report here the characterization of a gene,
DYX1C1
near the
DYX1
locus in chromosome 15q21, that is disrupted by a translocation t(2;15)(q11;q21) segregating coincidentally with dyslexia. Two sequence changes in
DYX1C1
, one involving the translation initiation sequence and an Elk-1 transcription factor binding site (–3G → A) and a codon (1249G → T), introducing a premature stop codon and truncating the predicted protein by 4 aa, associate alone and in combination with dyslexia.
DYX1C1
encodes a 420-aa protein with three tetratricopeptide repeat (TPR) domains, thought to be protein interaction modules, but otherwise with no homology to known proteins. The mouse Dyx2016 protein is 78% identical to the human protein, and the nonhuman primates differ at 0.5–1.4% of residues.
DYX1C1
is expressed in several tissues, including the brain, and the protein resides in the nucleus. In human brain, DYX1C1 protein localizes to a fraction of cortical neurons and white matter glial cells. We conclude that
DYX1C1
should be regarded as a candidate gene for developmental dyslexia. Detailed study of its function may open a path to understanding a complex process of development and maturation of the human brain.
Activation of ATP-sensitive K+(KATP) channels by H2O2underlies glutamate-dependent inhibition of striatal dopamine release
Authors: Avshalumov Marat V. , Rice Margaret E.
Published: October 2003

In many cells, ATP-sensitive K+channels (KATPchannels) couple metabolic state to excitability. In pancreatic beta cells, for example, this coupling regulates insulin release. Although KATPchannels are abundantly expressed in the brain, their physiological role and the factors that regulate them are poorly understood. One potential regulator is H2O2. We reported previously that dopamine (DA) release in the striatum is modulated by endogenous H2O2, generated downstream from glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor activation. Here we investigated whether H2O2-sensitive KATPchannels contribute to DA-release modulation by glutamate and γ-aminobutyric acid (GABA). This question is important because DA–glutamate interactions underlie brain functions, including motor control and cognition. Synaptic DA release was evoked by using local electrical stimulation in slices of guinea pig striatum and monitored in real time with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. The KATP-channel antagonist glibenclamide abolished the H2O2-dependent increase in DA release usually seen with AMPA-receptor blockade by GYKI-52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride] and the decrease in DA release seen with GABA-type-A-receptor blockade by picrotoxin. In contrast, 5-hydroxydecanoate, a mitochondrial KATP-channel blocker, was ineffective, as were sulpiride, a D2-receptor antagonist, and tertiapin, a G protein-coupled K+-channel inhibitor. Diazoxide, a sulfonylurea receptor 1 (SUR1)selective KATP-channel opener, prevented DA modulation by H2O2, glutamate, and GABA, whereas cromakalim, a SUR2-selective opener, did not. Thus, endogenous H2O2activates SUR1-containing KATPchannels in the plasma membrane to inhibit DA release. These data not only demonstrate that KATPchannels can modulate CNS transmitter release in response to fast-synaptic transmission but also introduce H2O2as a KATP-channel regulator.
Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid–detergent matrices
Authors: Paas Yoav , Cartaud Jean , Recouvreur Michel , Grailhe Regis , Dufresne Virginie , Pebay-Peyroula Eva , Landau Ehud M. , Changeux Jean-Pierre
Published: October 2003

Nicotinic acetylcholine receptors (AChRs) belong to a superfamily of oligomeric proteins that transduce electric signals across the cell membrane on binding of neurotransmitters. These receptors harbor a large extracellular ligand-binding domain directly linked to an ion-conducting channel-forming domain that spans the cell membrane 20 times and considerably extends into the cytoplasm. Thus far, none of these receptor channels has been crystallized in three dimensions. The crystallization of the AChR fromTorpedo marmorataelectric organs is challenged here in lipidic–detergent matrices. Detergent-soluble AChR complexed with α-bungarotoxin (αBTx), a polypeptidic competitive antagonist, was purified. The AChR–αBTx complex was reconstituted in a lipidic matrix composed of monoolein bilayers that are structured in three dimensions. The αBTx was conjugated to a photo-stable fluorophore, enabling us to monitor the physical behavior of the receptor–toxin complex in the lipidic matrix under light stereomicroscope, and to freeze fracture regions containing the receptor–toxin complex for visualization under a transmission electron microscope. Conditions were established for forming 2D receptor–toxin lattices that are stacked in the third dimension. 3D AChR nanocrystals were thereby grown inside the highly viscous lipidic 3D matrix. Slow emulsification of the lipidic matrix converted these nanocrystals into 3D elongated thin crystal plates of micrometer size. The latter are stable in detergent-containing aqueous solutions and can currently be used for seeding and epitaxial growth, en route to crystals of appropriate dimensions for x-ray diffraction studies.
The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences
Authors: Sakai Akito , Claire Marisa St. , Faulk Kristina , Govindarajan Sugantha , Emerson Suzanne U. , Purcell Robert H. , Bukh Jens
Published: October 2003

The role of the hepatitis C virus (HCV) p7 protein in the virus life cycle is not known. Previous
in vitro
data indicated that this 63-aa polypeptide is located in the endoplasmic reticulum and has two transmembrane domains (TMDs) connected by a cytoplasmic loop; the amino- and carboxyl-terminal tails are oriented toward the endoplasmic reticulum lumen. Furthermore, recent
in vitro
studies suggested that HCV p7 could function as a virus-encoded ion channel. It might therefore be a relevant target for future drug development. We studied the role of HCV p7
in vivo
. Because HCV does not replicate efficiently in cell culture, we mutagenized p7 of an infectious genotype 1a cDNA clone and tested RNA transcripts of each mutant for infectivity in chimpanzees by intrahepatic transfection. Appropriate processing of mutant polypeptides was confirmed by studies in transfected mammalian cells. Mutants with deletions of all or part of p7 and a mutant with substitutions of two conserved residues in the cytoplasmic loop were not viable. Thus, p7 is essential for infectivity of HCV. A chimera in which the p7 of the 1a clone was replaced with p7 from an infectious genotype 2a clone also was not viable. This finding suggests a genotype-specific interaction between p7 and other genomic regions. To define which portions of p7 played the most significant role for this interaction, we tested three chimeras with the 1a backbone in which only specific domains of p7 had the 2a sequence. A p7 chimera with 2a tails and TMDs and the 1a cytoplasmic loop was not viable. A mutant with 2a tails and cytoplasmic loop and 1a TMDs also was not viable. However, a p7 chimera with 2a TMDs and cytoplasmic loop and 1a tails was viable. The transfected chimpanzee became viremic at week 2, and recovered viruses had the chimeric sequence. These data indicate that the amino- and/or carboxyl-terminal intraluminal tails of p7 contain sequences with genotype-specific function.
A natural mutation in the Tyk2 pseudokinase domain underlies altered susceptibility of B10.Q/J mice to infection and autoimmunity
Authors: Shaw Michael H. , Boyartchuk Victor , Wong Sandy , Karaghiosoff Marina , Ragimbeau Josiane , Pellegrini Sandra , Muller Mathias , Dietrich William F. , Yap George S.
Published: October 2003

The B10.Q/J strain of mice was serendipitously discovered to be highly susceptible to infection by the intracellular protozoan parasite,Toxoplasma gondiibut markedly resistant to induction of autoimmune arthritis. We have previously shown that the B10.Q/J phenotype is controlled by a single recessive locus and is associated with lymphocyte hyporesponsiveness to IL-12. Using genetic approaches, we have now localized the B10.Q/J locus to chromosome 9 and established its identity asTyk2, a Janus kinase essential for IL-12 and IFN-α/β cytokine signaling. The B10.Q/JTyk2gene contained a single missense mutation resulting in a nonconservative amino acid substitution (E775K) in an invariant motif of the pseudokinase (Janus kinase homology 2) domain. This mutation appeared to result in the absence of the B10.Q/J-encoded Tyk2 protein, despite presence ofTyk2-specific transcripts. Phenotypically, B10.Q/J cells were indistinguishable fromTyk2-deficient cells, showing impaired signaling and biologic responses to IL-12, IL-23, and type I IFNs. The analogous E782K mutant of human Tyk2 failed to restore IFN-α responsiveness inTyk2null 11,1 cells. Our results indicate a crucial role for Tyk2 in T helper 1-mediated protective and pathogenic immune responses. An additional implication of our findings is that naturally occurring mutations in theTyk2gene may underlie altered susceptibilities to infectious or autoimmune diseases in human and animal populations.
Insulin regulates the activity of forkhead transcription factor Hnf-3β/Foxa-2 by Akt-mediated phosphorylation and nuclear/cytosolic localization
Authors: Wolfrum Christian , Besser Daniel , Luca Edlira , Stoffel Markus
Published: October 2003

Hepatocyte nuclear factors 3 α, β, and γ (Foxa-1, -2, and -3) are transcriptional activators of important metabolic genes in the liver that are suppressed by the actions of insulin. Here, we show that the activation of phosphatidylinositol 3-kinase–Akt by insulin induces Foxa-2 phosphorylation, nuclear exclusion, and inhibition of Foxa-2-dependent transcriptional activity. Foxa-2 physically interacts with Akt, a key mediator of the phosphatidylinositol 3-kinase pathway and is phosphorylated at a single conserved site (T156) that is absent in Foxa-1 and Foxa-3 proteins. This Akt phosphorylation site in Foxa-2 is highly conserved from mammals to insects. Mutant Foxa-2
T156A
is resistant to Akt-mediated phosphorylation, nuclear exclusion, and transcriptional inactivation of Foxa-2-regulated gene expression. These results implicate an evolutionarily conserved mechanism in the regulation of Foxa-2-dependent transcriptional control by extracellular signals such as insulin.
Mathematical models of RNA silencing: Unidirectional amplification limits accidental self-directed reactions
Authors: Bergstrom Carl T. , McKittrick Erin , Antia Rustom
Published: October 2003

RNA silencing, found broadly throughout the eukaryotes, posttranscriptionally suppresses the expression of “aberrant” genes including those of many viruses and transposons. Similar to the specific immune system of vertebrates, RNA silencing works by generating specific responses against foreign elements and rapidly amplifying these responses to clear or otherwise inactivate the threat. Also like the vertebrate immune system, RNA-silencing systems risk making mistakes and mounting undesirable responses against the self. We develop a set of mathematical models of RNA silencing. We show that current models of RNA silencing do little to explain what prevents mistaken reactions from silencing vital organismal genes. We extend the basic models to show that the presumed unidirectional nature of the amplification process (namely, unidirectional RNA-directed RNA polymerase-mediated synthesis of secondary double-stranded RNA as observed in
Caenorhabditis elegans
) serves as a “safety mechanism” that safeguards against accidental generation of damaging self-directed reactions.
A
1
adenosine receptors mediate hypoxia-induced ventriculomegaly
Authors: Turner Christopher P. , Seli Meltem , Ment Laura , Stewart William , Yan Henglin , Johansson Björn , Fredholm Bertil B. , Blackburn Michael , Rivkees Scott A.
Published: October 2003

Periventricular leukomalacia is characterized by a reduction in brain matter and secondary ventriculomegaly and is a major cause of developmental delay and cerebral palsy in prematurely born infants. Currently, our understanding of the pathogenesis of this condition is limited. In animal models, features of periventricular leukomalacia can be induced by hypoxia and activation of A
1
adenosine receptors (A
1
ARs). Using mice that are deficient in the A
1
AR gene (

A
1
AR

–/–), we show that A
1
ARs play a prominent role in the development of hypoxia-induced ventriculomegaly in neonates. Supporting a role for adenosine in the pathogenesis of developmental brain injury, ventriculomegaly was also observed in mice lacking the enzyme adenosine deaminase, which degrades adenosine. Thus, adenosine acting on A
1
ARs appears to mediate hypoxia-induced brain injury ventriculomegaly during early postnatal development.
Evolution's cauldron: Duplication, deletion, and rearrangement in the mouse and human genomes
Authors: Kent W. James , Baertsch Robert , Hinrichs Angie , Miller Webb , Haussler David
Published: October 2003

This study examines genomic duplications, deletions, and rearrangements that have happened at scales ranging from a single base to complete chromosomes by comparing the mouse and human genomes. From whole-genome sequence alignments, 344 large (>100-kb) blocks of conserved synteny are evident, but these are further fragmented by smaller-scale evolutionary events. Excluding transposon insertions, on average in each megabase of genomic alignment we observe two inversions, 17 duplications (five tandem or nearly tandem), seven transpositions, and 200 deletions of 100 bases or more. This includes 160 inversions and 75 duplications or transpositions of length >100 kb. The frequencies of these smaller events are not substantially higher in finished portions in the assembly. Many of the smaller transpositions are processed pseudogenes; we define a “syntenic” subset of the alignments that excludes these and other small-scale transpositions. These alignments provide evidence that ≈2% of the genes in the human/mouse common ancestor have been deleted or partially deleted in the mouse. There also appears to be slightly less nontransposon-induced genome duplication in the mouse than in the human lineage. Although some of the events we detect are possibly due to misassemblies or missing data in the current genome sequence or to the limitations of our methods, most are likely to represent genuine evolutionary events. To make these observations, we developed new alignment techniques that can handle large gaps in a robust fashion and discriminate between orthologous and paralogous alignments.
Calsequestrin determines the functional size and stability of cardiac intracellular calcium stores: Mechanism for hereditary arrhythmia
Authors: Terentyev Dmitry , Viatchenko-Karpinski Serge , Györke Inna , Volpe Pompeo , Williams Simon C. , Györke Sandor
Published: October 2003

Calsequestrin is a high-capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR), an intracellular Ca release and storage organelle in muscle. Mutations in the cardiac calsequestrin gene (
CSQ2
) have been linked to arrhythmias and sudden death. We have used Ca-imaging and patch-clamp methods in combination with adenoviral gene transfer strategies to explore the function of CSQ2 in adult rat heart cells. By increasing or decreasing CSQ2 levels, we showed that CSQ2 not only determines the Ca storage capacity of the SR but also positively controls the amount of Ca released from this organelle during excitation–contraction coupling. CSQ2 controls Ca release by prolonging the duration of Ca fluxes through the SR Ca-release sites. In addition, the dynamics of functional restitution of Ca-release sites after Ca discharge were prolonged when CSQ2 levels were elevated and accelerated in the presence of lowered CSQ2 protein levels. Furthermore, profound disturbances in rhythmic Ca transients in myocytes undergoing periodic electrical stimulation were observed when CSQ2 levels were reduced. We conclude that CSQ2 is a key determinant of the functional size and stability of SR Ca stores in cardiac muscle. CSQ2 appears to exert its effects by influencing the local luminal Ca concentration-dependent gating of the Ca-release channels and by acting as both a reservoir and a sink for Ca in SR. The abnormal restitution of Ca-release channels in the presence of reduced CSQ2 levels provides a plausible explanation for ventricular arrhythmia associated with mutations of
CSQ2
.
LAX
and
SPA
: Major regulators of shoot branching in rice
Authors: Komatsu Keishi , Maekawa Masahiko , Ujiie Shin , Satake Yuzuki , Furutani Ikuyo , Okamoto Hironobu , Shimamoto Ko , Kyozuka Junko
Published: October 2003

The aerial architecture of plants is determined primarily by the pattern of shoot branching. Although shoot apical meristem initiation during embryogenesis has been extensively studied by molecular genetic approaches using
Arabidopsis
, little is known about the genetic mechanisms controlling axillary meristem initiation, mainly because of the insufficient number of mutants that specifically alter it. We identified the
LAX PANICLE
(
LAX
) and
SMALL PANICLE
(
SPA
) genes as the main regulators of axillary meristem formation in rice.
LAX
encodes a basic helix–loop–helix transcription factor and is expressed in the boundary between the shoot apical meristem and the region of new meristem formation. This pattern of
LAX
expression was repeatedly observed in every axillary meristem, consistent with our observation that
LAX
is involved in the formation of all types of axillary meristems throughout the ontogeny of a rice plant. Ectopic
LAX
expression in rice caused pleiotropic effects, including dwarfing, an altered pattern of stem elongation, darker color, bending of the lamina joint, absence of the midribs of leaves, and severe sterility.
A noncognate aminoacyl-tRNA synthetase that may resolve a missing link in protein evolution
Authors: Skouloubris Stéphane , de Pouplana Lluís Ribas , de Reuse Hilde , Hendrickson Tamara L.
Published: October 2003

Efforts to delineate the advent of many enzymes essential to protein translation are often limited by the fact that the modern genetic code evolved before divergence of the tree of life. Glutaminyl-tRNA synthetase (GlnRS) is one noteworthy exception to the universality of the translation apparatus. In eukaryotes and some bacteria, this enzyme is essential for the biosynthesis of Gln-tRNA
Gln
, an obligate intermediate in translation. GlnRS is absent, however, in archaea, and most bacteria, organelles, and chloroplasts. Phylogenetic analyses predict that GlnRS arose from glutamyl-tRNA synthetase (GluRS), via gene duplication with subsequent evolution of specificity. A pertinent question to ask is whether, in the advent of GlnRS, a transient GluRS-like intermediate could have been retained in an extant organism. Here, we report the discovery of an essential GluRS-like enzyme (GluRS2), which coexists with another GluRS (GluRS1) in
Helicobacter pylori
. We show that GluRS2's primary role is to generate Glu-tRNA
Gln
, not Glu-tRNA
Glu
. Thus, GluRS2 appears to be a transient GluRS-like ancestor of GlnRS and can be defined as a GluGlnRS.
Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter
Authors: Fanucci Gail E. , Cadieux Nathalie , Kadner Robert J. , Cafiso David S.
Published: October 2003

BtuB is a TonB-dependent outer-membrane transporter for vitamin B
12
(or cyanocobalamin, CN-Cbl) in
Escherichia coli
. The binding of CN-Cbl is believed to promote an unfolding or undocking of the Ton box, the conserved N-terminal energy coupling motif at the periplasmic surface of the transporter. This structural change may facilitate the interaction of BtuB with the inner membrane protein TonB. In this work, the effect of the receptor binding fragment of colicin E3 (E3R) on the conformation of the Ton box was examined with site-directed spin labeling. Addition of E3R reverses the undocking of the Ton box that is promoted by CN-Cbl, consistent with a competitive binding between the substrate and the colicin fragment. EPR spectroscopy indicates that the Ton box is in a two-state equilibrium between docked and undocked conformations. In the absence of substrate, the docked conformation is the predominant state; however, the equilibrium can be shifted to the undocked state by the addition of detergents or site-specific proline substitutions. Even when the undocking is induced by detergents or by certain proline mutations, E3R binding shifts the equilibrium to the docked conformation. Thus, two competitive extracellular ligands, CN-Cbl and ER3, transduce opposite conformations of the N-terminal Ton box. Substrate binding stabilizes an undocked conformation, whereas E3R binding stabilizes a docked conformation of the Ton box.
Natural variation in nucleolar dominance reveals the relationship between nucleolus organizer chromatin topology and rRNA gene transcription in
Arabidopsis
Authors: Pontes Olga , Lawrence Richard J. , Neves Nuno , Silva Manuela , Lee Jae-Hyeok , Chen Z. Jeffrey , Viegas Wanda , Pikaard Craig S.
Published: October 2003

In genetic hybrids, nucleolus formation on chromosomes inherited from only one parent is the epigenetic phenomenon, nucleolar dominance. By using
Arabidopsis suecica
, the allotetraploid hybrid of
Arabidopsis thaliana
and
Arabidopsis arenosa
, natural variation in nucleolar dominance was found to occur, providing a unique opportunity to examine homologous nucleolus organizer regions (NORs) in their active and inactive states. In
A. suecica
strain LC1, NORs derived from
A. arenosa
are active, whereas
A. thaliana
-derived NORs are silenced. In
A. suecica
strain 9502, NORs of both parental species are active. When active, NORs are partially, but not fully, decondensed. Both active and inactive LC1 NORs colocalize with the nucleolus, contradicting the long-standing assumption that rRNA gene transcription drives nucleolus association. Collectively, these observations clarify the relationships among NOR chromatin topology, rRNA gene transcription, and NOR–nucleolus associations.
A. suecica
strains LC1 and 9502 have each lost one pair of
A. thaliana
NORs during evolution, and amplified fragment-length polymorphism analysis further indicates that these strains are genetically very similar. These data suggest that nucleolar dominance can result from subtle genetic or epigenetic variation but is not a trait fundamental to a given interspecies hybrid combination.
Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection
Authors: Rausch Jason W. , Qu Jin , Yi-Brunozzi Hye Young , Kool Eric T. , Le Grice Stuart F. J.
Published: October 2003

Both x-ray crystallography and chemical footprinting indicate that bases of the HIV type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson–Crick base pairing. However, the contribution of these structural anomalies to the accuracy of plus-strand primer selection by HIV-1 reverse transcriptase is not immediately clear. To address this issue, DNA templates harboring single and pairwise non-hydrogen-bonding isosteres of cytosine (2-fluoro-4-methylbenzene deoxyribonucleoside) and thymine (2,4-difluoro-5-methylbenzene deoxyribonucleoside) were synthesized and hybridized to PPT-containing RNA primers as a means of locally removing hydrogen bonding and destabilizing paired structure. Cleavage of these hybrids was examined with p66/p51 HIV-1 reverse transcriptase and a mutant carrying an alteration in the p66 RNase H primer shown to specifically impair PPT processing. Analog insertion within the PPT (rG):(dC) and central (rA):(dT) tracts repositioned the RNase H domain such that the RNA/DNA hybrid was cleaved 3–4 bp from the site of insertion, a distance corresponding closely to the spatial separation between the catalytic center and RNase H primer grip. However, PPT processing was significantly impaired when the junction between these tracts was substituted. Substitutions within the upstream (rA):(dT) tract, where maximum distortion had previously been observed, destroyed PPT processing. Collectively, our scanning mutagenesis approach implicates multiple regions of the PPT in the accuracy with which it is excised from (+) U3 RNA and DNA, and also provides evidence for close cooperation between the RNase H primer grip and catalytic center in achieving this cleavage.
Calcification in atherosclerosis: Bone biology and chronic inflammation at the arterial crossroads
Authors: Doherty Terence M. , Asotra Kamlesh , Fitzpatrick Lorraine A. , Qiao Jian-Hua , Wilkin Douglas J. , Detrano Robert C. , Dunstan Colin R. , Shah Prediman K. , Rajavashisth Tripathi B.
Published: October 2003

Dystrophic or ectopic mineral deposition occurs in many pathologic conditions, including atherosclerosis. Calcium mineral deposits that frequently accompany atherosclerosis are readily quantifiable radiographically, serve as a surrogate marker for the disease, and predict a higher risk of myocardial infarction and death. Accelerating research interest has been propelled by a clear need to understand how plaque structure, composition, and stability lead to devastating cardiovascular events. In atherosclerotic plaque, accumulating evidence is consistent with the notion that calcification involves the participation of arterial osteoblasts and osteoclasts. Here we summarize current models of intimal arterial plaque calcification and highlight intriguing questions that require further investigation. Because atherosclerosis is a chronic vascular inflammation, we propose that arterial plaque calcification is best conceptualized as a convergence of bone biology with vascular inflammatory pathobiology.
Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine 4
Authors: Morshead Katrina B. , Ciccone David N. , Taverna Sean D. , Allis C. David , Oettinger Marjorie A.
Published: October 2003

In the earliest stages of antigen receptor assembly, D and J segments of the Ig heavy chain and T cell receptor β loci are recombined in B and T cells, respectively, whereas the V segments are not. Distinct distribution patterns of various histone modifications and the nucleosome-remodeling factor BRG1 are found at “active” (DJ) and “inactive” (V) regions. Striking “hotspots” of histone H3 dimethylated at lysine 4 (di-Me H3-K4) are localized at the ends of the active DJ domains of both the Ig heavy chain and T cell receptor β loci. BRG1 is not localized to specific sequences, as it is with transcriptional initiation, but rather associates with the entire active locus in a pattern that mirrors acetylation of histone H3. Within some inactive loci marked by H3-K9 dimethylation, two distinct levels of methylation are found in a nonrandom gene-segment-specific pattern. We suggest that the hotspots of di-Me H3-K4 are important marks for locus accessibility. The specific patterns of modification imply that the regulation of V(D)J recombination involves recruitment of specific methyltransferases in a localized manner.
Dynamics of feature binding during object-selective attention
Authors: Schoenfeld M. A. , Tempelmann C. , Martinez A. , Hopf J.-M. , Sattler C. , Heinze H.-J. , Hillyard S. A.
Published: October 2003

Objects in the environment may be attended selectively and perceived as unified ensembles of their constituent features. To investigate the timing and cortical localization of feature-integration mechanisms in object-based attention, recordings of event-related potentials and magnetic fields were combined with functional MRI while subjects attended to one of two superimposed transparent surfaces formed by arrays of dots moving in opposite directions. A spatiotemporal analysis revealed evidence for a rapid increase in neural activity localized to a color-selective region of the fusiform gyrus when the surface moving in the attended direction displayed an irrelevant color feature. These data provide support for the “integrated-competition” model of object-selective attention and point to a dynamic neural substrate for the rapid binding process that links relevant and irrelevant features to form a unified perceptual object.