bluuebunny/crossref_metadata_2025 huggingface dataset:
Working memory and fear conditioning
Authors: Carter Ronald McKell , Hofstötter Constanze , Tsuchiya Naotsugu , Koch Christof
Published: February 2003

Previous studies of associative learning implicate higher-level cognitive processes in some forms of classical conditioning. An ongoing debate is concerned with the extent to which attention and awareness are necessary for trace but not delay eye-blink conditioning [Clark, R. E. & Squire, L. R. (1998)
Science
280, 77–81; Lovibond, P. F. & Shanks, D. (2002)
J. Exp. Psychol. Anim. Behav. Processes
28, 38–42]. In trace conditioning, a short interval is interposed between the termination of the conditioned stimulus (CS) and the onset of the unconditioned stimulus (US). In delay conditioning, the CS and US overlap. We here investigate the extent to which human classical fear conditioning depends on working memory. Subjects had to carry out an
n
-back task, requiring tracking an item 1 or 2 back in a sequentially presented list of numbers, while simultaneously being tested for their ability to associate auditory cues with shocks under a variety of conditions (single-cue versus differential; delay versus trace; no task versus 0-, 1-, and 2-back). Differential delay conditioning proved to be more resilient than differential trace conditioning but does show a reduction due to task interference similar in slope to that found in trace conditioning. Explicit knowledge of the stimulus contingency facilitates but does not guarantee trace conditioning. Only the single-cue delay protocol shows conditioning during the more difficult working memory task. Our findings suggest that the larger the cognitive demands on the system, the less likely conditioning occurs. A postexperimental questionnaire showed a positive correlation between conditioning and awareness for differential trace conditioning extinction.
Parallel changes in gene expression after 20,000 generations of evolution inEscherichiacoli
Authors: Cooper Tim F. , Rozen Daniel E. , Lenski Richard E.
Published: February 2003

Twelve populations ofEscherichia coli, derived from a common ancestor, evolved in a glucose-limited medium for 20,000 generations. Here we use DNA expression arrays to examine whether gene-expression profiles in two populations evolved in parallel, which would indicate adaptation, and to gain insight into the mechanisms underlying their adaptation. We compared the expression profile of the ancestor to that of clones sampled from both populations after 20,000 generations. The expression of 59 genes had changed significantly in both populations. Remarkably, all 59 were changed in the same direction relative to the ancestor. Many of these genes were members of the cAMP-cAMP receptor protein (CRP) and guanosine tetraphosphate (ppGpp) regulons. Sequencing of several genes controlling the effectors of these regulons found a nonsynonymous mutation inspoTin one population. Moving this mutation into the ancestral background showed that it increased fitness and produced many of the expression changes manifest after 20,000 generations. The same mutation had no effect on fitness when introduced into the other evolved population, indicating that a mutation of similar effect was present already. Our study demonstrates the utility of expression arrays for addressing evolutionary issues including the quantitative measurement of parallel evolution in independent lineages and the identification of beneficial mutations.
Competition for self ligands restrains homeostatic proliferation of naive CD4 T cells
Authors: Moses Christina T. , Thorstenson Kristen M. , Jameson Stephen C. , Khoruts Alexander
Published: February 2003

T cell antigen receptor (TCR) diversity is a critical feature of adaptive immunity. However, restriction of TCR diversity is a potential risk during immune reconstitution by homeostatic proliferation. What peripheral mechanisms are in place to maintain TCR diversity during recovery from lymphopenia? Here, we examine competition between several monoclonal CD4 T cell populations in RAG−/−and TCR Tg RAG−/−environments. The results suggest that specific self ligands constitute a critical limiting resource essential for homeostatic proliferation of naive CD4 T cells. In addition, T cells ignore large numbers of competitors as long as their TCR specificity is different and other non-MHC resources are not limiting. Therefore, the numbers of self ligands expressed in the periphery set the limits on TCR diversity.
An evolutionary scenario for the transition to undifferentiated multicellularity
Authors: Pfeiffer Thomas , Bonhoeffer Sebastian
Published: February 2003

The evolutionary transition from single cells toward multicellular forms of life represents one of the major transitions in the evolution of complex organisms. In this transition, single autonomously reproducing cells became parts of larger reproducing entities that eventually constituted a new unit of selection. The first step in the evolutionary transition to multicellularity likely was the evolution of simple, undifferentiated cell clusters. However, what the selective advantage of such cell clusters may have been remains unclear. Here, we argue that in populations of unicellular organisms with cooperative behavior, clustering may be beneficial by reducing interactions with noncooperative individuals. In support of this hypothesis, we present a set of computer simulations showing that clustering can evolve as a biological, heritable trait for cells that cooperate in the use of external energy resources. Following the evolution of simple cell clusters, further benefits could have arisen from the exchange of resources between cells of a cluster.
Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype
Authors: Yang F. , Alkalaeva E. Z. , Perelman P. L. , Pardini A. T. , Harrison W. R. , O'Brien P. C. M. , Fu B. , Graphodatsky A. S. , Ferguson-Smith M. A. , Robinson T. J.
Published: February 2003

The Afrotheria, a supraordinal grouping of mammals whose radiation is rooted in Africa, is strongly supported by DNA sequence data but not by their disparate anatomical features. We have used flow-sorted human, aardvark, and African elephant chromosome painting probes and applied reciprocal painting schemes to representatives of two of the Afrotherian orders, the Tubulidentata (aardvark) and Proboscidea (elephants), in an attempt to shed additional light on the evolutionary affinities of this enigmatic group of mammals. Although we have not yet found any unique cytogenetic signatures that support the monophyly of the Afrotheria, embedded within the aardvark genome we find the strongest evidence yet of a mammalian ancestral karyotype comprising 2
n
= 44. This karyotype includes nine chromosomes that show complete conserved synteny to those of man, six that show conservation as single chromosome arms or blocks in the human karyotype but that occur on two different chromosomes in the ancestor, and seven neighbor-joining combinations (i.e., the synteny is maintained in the majority of species of the orders studied so far, but which corresponds to two chromosomes in humans). The comparative chromosome maps presented between human and these Afrotherian species provide further insight into mammalian genome organization and comparative genomic data for the Afrotheria, one of the four major evolutionary clades postulated for the Eutheria.
Plant-like traits associated with metabolism of
Trypanosoma
parasites
Authors: Hannaert Véronique , Saavedra Emma , Duffieux Francis , Szikora Jean-Pierre , Rigden Daniel J. , Michels Paul A. M. , Opperdoes Fred R.
Published: February 2003

Trypanosomatid parasites cause serious diseases among humans, livestock, and plants. They belong to the order of the Kinetoplastida and form, together with the Euglenida, the phylum Euglenozoa. Euglenoid algae possess plastids capable of photosynthesis, but plastids are unknown in trypanosomatids. Here we present molecular evidence that trypanosomatids possessed a plastid at some point in their evolutionary history. Extant trypanosomatid parasites, such as
Trypanosoma
and
Leishmania
, contain several “plant-like” genes encoding homologs of proteins found in either chloroplasts or the cytosol of plants and algae. The data suggest that kinetoplastids and euglenoids acquired plastids by endosymbiosis before their divergence and that the former lineage subsequently lost the organelle but retained numerous genes. Several of the proteins encoded by these genes are now, in the parasites, found inside highly specialized peroxisomes, called glycosomes, absent from all other eukaryotes, including euglenoids.
Eye opening induces a rapid dendritic localization of PSD-95 in central visual neurons
Authors: Yoshii Akira , Sheng Morgan H. , Constantine-Paton Martha
Published: February 2003

The membrane-associated guanylate kinase PSD-95 scaffolds
N
-methyl-
d
-aspartate receptors to cytoplasmic signaling molecules, and associates with other glutamate receptors at central synapses. However, regulation of PSD-95
in vivo
is poorly understood. We provide evidence of an activity-dependent redistribution of PSD-95 to dendrites in central visual neurons that is tied to eye opening. Six hours after eye opening, increased dendritic PSD-95 coimmunoprecipitates with the same proportions of stargazin, increased proportions of the
N
-methyl-
d
-aspartate receptor subunit NR2A, and decreased proportions of NR2B. Sustained high levels of PSD-95 in dendrites are dependent on continued pattern vision in juvenile but not mature animals, suggesting that the stabilization of PSD-95 at synapses may be involved in the control of developmental plasticity.
Least effort and the origins of scaling in human language
Authors: Cancho Ramon Ferrer i , Solé Ricard V.
Published: February 2003

The emergence of a complex language is one of the fundamental events of human evolution, and several remarkable features suggest the presence of fundamental principles of organization. These principles seem to be common to all languages. The best known is the so-called Zipf's law, which states that the frequency of a word decays as a (universal) power law of its rank. The possible origins of this law have been controversial, and its meaningfulness is still an open question. In this article, the early hypothesis of Zipf of a principle of least effort for explaining the law is shown to be sound. Simultaneous minimization in the effort of both hearer and speaker is formalized with a simple optimization process operating on a binary matrix of signal–object associations. Zipf's law is found in the transition between referentially useless systems and indexical reference systems. Our finding strongly suggests that Zipf's law is a hallmark of symbolic reference and not a meaningless feature. The implications for the evolution of language are discussed. We explain how language evolution can take advantage of a communicative phase transition.
Unique mitochondrial genome architecture in unicellular relatives of animals
Authors: Burger Gertraud , Forget Lise , Zhu Yun , Gray Michael W. , Lang B. Franz
Published: February 2003

Animal mtDNAs are typically small (≈16 kbp), circular-mapping molecules that encode 37 or fewer tightly packed genes. Here we investigate whether similarly compact mitochondrial genomes are also present in the closest unicellular relatives of animals, i.e., choanoflagellate and ichthyosporean protists. We find that the gene content and architecture of the mitochondrial genomes of the choanoflagellate
Monosiga
brevicollis
, the ichthyosporean
Amoebidium
parasiticum
, and Metazoa are radically different from one another. The circular-mapping choanoflagellate mtDNA with its long intergenic regions is four times as large and contains two times as many protein genes as do animal mtDNAs, whereas the ichthyosporean mitochondrial genome totals >200 kbp and consists of several hundred linear chromosomes that share elaborate terminal-specific sequence patterns. The highly peculiar organization of the ichthyosporean mtDNA raises questions about the mechanism of mitochondrial genome replication and chromosome segregation during cell division in this organism. Considering that the closest unicellular relatives of animals possess large, spacious, gene-rich mtDNAs, we posit that the distinct compaction characteristic of metazoan mitochondrial genomes occurred simultaneously with the emergence of a multicellular body plan in the animal lineage.
Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor
Authors: Chauhan Sharmila D. , Nilsson Holger , Ahluwalia Amrita , Hobbs Adrian J.
Published: February 2003

Endothelial cells in most vascular beds release a factor that hyperpolarizes the underlying smooth muscle, produces vasodilatation, and plays a fundamental role in the regulation of local blood flow and systemic blood pressure. The identity of this endothelium-derived hyperpolarizing factor (EDHF), which is neither NO nor prostacyclin, remains obscure. Herein, we demonstrate that in mesenteric resistance arteries, release of C-type natriuretic peptide (CNP) accounts for the biological activity of EDHF. Both produce identical smooth muscle hyperpolarizations that are attenuated in the presence of high [K
+
], the G
i
G protein (G
i
) inhibitor pertussis toxin, the G protein-gated inwardly rectifying K
+
channel inhibitor tertiapin, and a combination of Ba
2+
(inwardly rectifying K
+
channel blocker) plus ouabain (Na
+
/K
+
-ATPase inhibitor). Responses to EDHF and CNP are unaffected by the natriuretic peptide receptor (NPR)-A/B antagonist HS-142-1, but mimicked by the selective NPR-C agonist, cANF
4–23
. EDHF-dependent relaxation is concomitant with liberation of endothelial CNP; in the presence of the myoendothelial gap-junction inhibitor 18α-glycyrrhetinic acid or after endothelial denudation, CNP release and EDHF responses are profoundly suppressed. These data demonstrate that acetylcholine-evoked release of endothelial CNP activates NPR-C on vascular smooth muscle that via a G
i
coupling promotes Ba
2+
/ouabain-sensitive hyperpolarization. Thus, we have revealed the identity of EDHF and established a pivotal role for endothelial-derived CNP in the regulation of vascular tone and blood flow.
Innate and acquired immunity intersect in a global view of the acute-phase response
Authors: Yoo Joo-Yeon , Desiderio Stephen
Published: February 2003

Recognition of stereotypic chemical patterns by sentinel cells of the innate immune system provokes a transient deviation from homeostasis, the acute-phase response (APR). Although APR effectors have been identified individually, the complexity of the response suggested that emergent properties would be uncovered by a more comprehensive examination. Our global assessment revealed that ≈7% of genes in the mouse are mobilized in the hepatic APR to endotoxin. Extensive metabolic adjustments include suppression of pathways for cholesterol, fatty acid, and phospholipid synthesis. Increased expression of genes for innate defense was accompanied by coordinate induction of the MHC class I antigen presentation machinery, illustrating an intersection between innate and adaptive immunity.
Components of the Rb pathway are critical targets of UV mutagenesis in a murine melanoma model
Authors: Kannan Karuppiah , Sharpless Norman E. , Xu Jin , O'Hagan Ronan C. , Bosenberg Marcus , Chin Lynda
Published: February 2003

Epidemiological studies support a link between melanoma risk and UV exposure early in life, yet the molecular targets of UV's mutagenic actions are not known. By using well characterized murine models of melanoma, we provide genetic and molecular evidence that identifies components of the Rb pathway as the principal targets of UV mutagenesis in murine melanoma development. In a melanoma model driven by H-RAS activation and loss of p19
ARF
function, UV exposure resulted in a marked acceleration in melanoma genesis, with nearly half of these tumors harboring amplification of
cyclin-dependent kinase
(
cdk
)
6
, whereas none of the melanomas arising in the absence of UV treatment possessed
cdk6
amplification. Moreover, UV-induced melanomas showed a strict reciprocal relationship between
cdk6
amplification and p16
INK4a
loss, which is consistent with the actions of UV along the Rb pathway. Most significantly, UV exposure had no impact on the kinetics of melanoma driven by H-RAS activation and p16
INK4a
deficiency. Together, these molecular and genetic data identify components of the Rb pathway as critical biological targets of UV-induced mutagenesis in the development of murine melanoma
in vivo
.
Transplanted bone marrow generates new neurons in human brains
Authors: Mezey Éva , Key Sharon , Vogelsang Georgia , Szalayova Ildiko , Lange G. David , Crain Barbara
Published: February 2003

Adult bone marrow stem cells seem to differentiate into muscle, skin, liver, lung, and neuronal cells in rodents and have been shown to regenerate myocardium, hepatocytes, and skin and gastrointestinal epithelium in humans. Because we have demonstrated previously that transplanted bone marrow cells can enter the brain of mice and differentiate into neurons there, we decided to examine postmortem brain samples from females who had received bone marrow transplants from male donors. The underlying diseases of the patients were lymphocytic leukemia and genetic deficiency of the immune system, and they survived between 1 and 9 months after transplant. We used a combination of immunocytochemistry (utilizing neuron-specific antibodies) and fluorescent
in situ
hybridization histochemistry to search for Y chromosome-positive cells. In all four patients studied we found cells containing Y chromosomes in several brain regions. Most of them were nonneuronal (endothelial cells and cells in the white matter), but neurons were certainly labeled, especially in the hippocampus and cerebral cortex. The youngest patient (2 years old), who also lived the longest time after transplantation, had the greatest number of donor-derived neurons (7 in 10,000). The distribution of the labeled cells was not homogeneous. There were clusters of Y-positive cells, suggesting that single progenitor cells underwent clonal expansion and differentiation. We conclude that adult human bone marrow cells can enter the brain and generate neurons just as rodent cells do. Perhaps this phenomenon could be exploited to prevent the development or progression of neurodegenerative diseases or to repair tissue damaged by infarction or trauma.
A critical role for PPARα-mediated lipotoxicity in the pathogenesis of diabetic cardiomyopathy: Modulation by dietary fat content
Authors: Finck Brian N. , Han Xianlin , Courtois Michael , Aimond Franck , Nerbonne Jeanne M. , Kovacs Attila , Gross Richard W. , Kelly Daniel P.
Published: February 2003

To explore the role of peroxisome proliferator-activated receptor α (PPARα)-mediated derangements in myocardial metabolism in the pathogenesis of diabetic cardiomyopathy, insulinopenic mice with PPARα deficiency (PPARα
−/−
) or cardiac-restricted overexpression [myosin heavy chain (MHC)-PPAR] were characterized. Whereas PPARα
−/−
mice were protected from the development of diabetes-induced cardiac hypertrophy, the combination of diabetes and the MHC-PPAR genotype resulted in a more severe cardiomyopathic phenotype than either did alone. Cardiomyopathy in diabetic MHC-PPAR mice was accompanied by myocardial long-chain triglyceride accumulation. The cardiomyopathic phenotype was exacerbated in MHC-PPAR mice fed a diet enriched in triglyceride containing long-chain fatty acid, an effect that was reversed by discontinuing the high-fat diet and absent in mice given a medium-chain triglyceride-enriched diet. Reactive oxygen intermediates were identified as candidate mediators of cardiomyopathic effects in MHC-PPAR mice. These results link dysregulation of the PPARα gene regulatory pathway to cardiac dysfunction in the diabetic and provide a rationale for serum lipid-lowering strategies in the treatment of diabetic cardiomyopathy.
Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia
Authors: Comerford Katrina M. , Leonard Martin O. , Karhausen Jorn , Carey Robyn , Colgan Sean P. , Taylor Cormac T.
Published: February 2003

Phosphorylation-dependent ubiquitination combined with proteasomal degradation of transcriptional regulators is a recently appreciated mechanism for control of a number of inflammatory genes. Far less is known about the counterregulatory mechanisms that repress transcriptional activity in these pathways during resolution. Here, we investigated the transient nature of hypoxia-induced tumor necrosis factor (TNF)α in T84 cells, a process we have previously shown to involve phosphorylation-dependent degradation of the cAMP-response element-binding protein (CREB). Initial studies indicate hypoxia-induced TNFα to be a transient event, the resolution of which is associated with the appearance of a higher molecular weight modified form of CREB. Gene array analysis of mRNA derived from hypoxic cells identified a time-dependent induction of small ubiquitin-related modifier (SUMO)-1 mRNA. In prolonged hypoxia, CREB is posttranslationally modified by SUMO-1. Furthermore, SUMO-1 overexpression stabilizes CREB in hypoxia and enhances CREB-dependent reporter gene activity. Site-directed mutagenesis of lysine residues K285 and K304 identifies them as SUMO acceptors
in vivo
and
in vitro
. Mutation of K304 also results in loss of CREB nuclear localization, implying a role for SUMO-1 modification at this site in the subcellular localization of CREB. Thus, in prolonged hypoxia, CREB is modified by association with SUMO-1. Furthermore, we hypothesize that such an event stabilizes and promotes nuclear localization of CREB and thus complements an endogenous resolution phase for hypoxia-induced inflammatory processes.
Genetic biases for showy males: Are some genetic systems especially conducive to sexual selection?
Authors: Reeve Hudson Kern , Pfennig David W.
Published: February 2003

Male secondary sexual characters (conspicuous ornaments, signals, colors) are among nature's most striking features. Yet, it is unclear why certain groups of organisms are more likely than others to evolve these traits. One explanation for such taxonomic biases is that some genetic systems may be especially conducive to sexual selection. Here, we present theory and simulation results demonstrating that rare alleles encoding either male ornaments or female preferences for those ornaments are better protected against random loss in species with ZZ/ZW or ZZ/ZO sex chromosome systems (male homogamety) than in species with XX/XY or XX/XO systems (male heterogamety). Moreover, this protection is much stronger in diploid than haplodiploid species. We also present empirical data showing that male secondary sexual characters are better developed in diploid than haplodiploid species and in diploid species with male homogamety than in those with male heterogamety. Thus, taxonomic biases for showy males may stem from differences in sex chromosome systems.
CCAAT/enhancer-binding protein β is required for mitotic clonal expansion during adipogenesis
Authors: Tang Qi-Qun , Otto Tamara C. , Lane M. Daniel
Published: February 2003

Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAAT/enhancer-binding protein (C/EBP)β is expressed immediately but gains DNA-binding activity only after a long lag as the cells synchronously begin mitotic clonal expansion (MCE). After MCE, a process required for adipogenesis, C/EBPβ activates expression of C/EBPα and peroxisome proliferator-activated receptor γ, which then transcriptionally activate genes that produce the adipocyte phenotype. When mouse embryo fibroblasts (MEFs) are subjected to the same differentiation protocol, a subset of the MEFs undergoes a similar program of events. Similar to 3T3-L1 preadipocytes, the MEFs reenter the cell cycle (as indicated by the synchronous expression of cyclin A) and undergo MCE as evidenced by the incorporation of BrdUrd into DNA and the formation of mitotic foci of cells that undergo adipogenesis. C/EBPβ is expressed immediately after induction but exhibits delayed acquisition of DNA-binding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from C/EBPβ(−/−) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of C/EBPβ (LAP) but not dominant-negative C/EBPβ (LIP) in C/EBPβ(−/−) MEFs restores MCE, expression of adipocyte markers, and the capacity to form mitotic foci of cells that undergo adipogenesis. These findings demonstrate that expression of C/EBPβ is a prerequisite for MCE in the adipocyte-differentiation program.
Lectin-like oxidized LDL receptor-1 is a cell-adhesion molecule involved in endotoxin-induced inflammation
Authors: Honjo Megumi , Nakamura Kayo , Yamashiro Kenji , Kiryu Junichi , Tanihara Hidenobu , McEvoy Leslie M. , Honda Yoshihito , Butcher Eugene C. , Masaki Tomoh , Sawamura Tatsuya
Published: February 2003

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a major endothelial receptor for oxidized low-density lipoprotein, and is assumed to play a proatherogenic role in atherosclerosis. LOX-1 expression is induced by inflammatory cytokines as well as by proatherogenic stimuli. LOX-1 protein binds aged/apoptotic cells, activated platelets, and bacteria, suggesting that it may have diverse activities
in vivo
. Here, we reveal a role for LOX-1 in endotoxin-induced inflammation. In a model of endotoxemia, injection of a high dose of endotoxin into rats induced leukopenia within 1 h and death of the animals within 24 h. Preadministration of anti-LOX-1 antibody reduced the degree of leukopenia and completely rescued the animals, whereas control IgG did not. In a model of low-dose endotoxin-induced uveitis, anti-LOX-1 antibody efficiently suppressed leukocyte infiltration and protein exudation.
In situ
videomicroscopic analyses of leukocyte interactions with retinal veins revealed that anti-LOX-1 antibody reduced the number of rolling leukocytes and increased the velocity of rolling, suggesting that LOX-1 functions as a vascular tethering ligand. The ability of LOX-1 to capture leukocytes under physiologic shear was confirmed in an
in vitro
flow model. Thus, LOX-1 is an adhesion molecule involved in leukocyte recruitment and may represent an attractive target for modulation of endotoxin-induced inflammation.
Comparison of mouse and human genomes followed by experimental verification yields an estimated 1,019 additional genes
Authors: Guigó Roderic , Dermitzakis Emmanouil T. , Agarwal Pankaj , Ponting Chris P. , Parra Genís , Reymond Alexandre , Abril Josep F. , Keibler Evan , Lyle Robert , Ucla Catherine , Antonarakis Stylianos E. , Brent Michael R.
Published: February 2003

A primary motivation for sequencing the mouse genome was to accelerate the discovery of mammalian genes by using sequence conservation between mouse and human to identify coding exons. Achieving this goal proved challenging because of the large proportion of the mouse and human genomes that is apparently conserved but apparently does not code for protein. We developed a two-stage procedure that exploits the mouse and human genome sequences to produce a set of genes with a much higher rate of experimental verification than previously reported prediction methods. RT-PCR amplification and direct sequencing applied to an initial sample of mouse predictions that do not overlap previously known genes verified the regions flanking one intron in 139 predictions, with verification rates reaching 76%. On average, the confirmed predictions show more restricted expression patterns than the mouse orthologs of known human genes, and two-thirds lack homologs in fish genomes, demonstrating the sensitivity of this dual-genome approach to hard-to-find genes. We verified 112 previously unknown homologs of known proteins, including two homeobox proteins relevant to developmental biology, an aquaporin, and a homolog of dystrophin. We estimate that transcription and splicing can be verified for >1,000 gene predictions identified by this method that do not overlap known genes. This is likely to constitute a significant fraction of the previously unknown, multiexon mammalian genes.
Activation of human eosinophils through leukocyte immunoglobulin-like receptor 7
Authors: Tedla Nicodemus , Bandeira-Melo Christianne , Tassinari Paolo , Sloane David E. , Samplaski Mary , Cosman David , Borges Luis , Weller Peter F. , Arm Jonathan P.
Published: February 2003

Eosinophils are implicated prominently in allergic diseases and the host response to parasitic infections. Eosinophils may be activatedin vitroby diverse classes of agonists such as immunoglobulins, lipid mediators, and cytokines. The leukocyte Ig-like receptors (LIRs) comprise a family of inhibitory and activating cell-surface receptors. Inhibitory LIRs down-regulate cellular responses through cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. There are limited data on the action of the activating LIRs, which are thought to signal through the Fc receptor γ chain, which contains an immunoreceptor tyrosine-based activation motif. We now demonstrate the expression of LIR1 (inhibitory), LIR2 (inhibitory), LIR3 (inhibitory), and LIR7 (activating) on eosinophils from 4, 4, 12, and 11, respectively, of 12 healthy donors. Cross-linking of LIR7 with plate-bound antibody elicited the dose- and time-dependent release of eosinophil-derived neurotoxin and leukotriene C4. Eosinophils activated with antibodies to LIR7 embedded in gel-phase EliCell preparations showed leukotriene C4generation at the nuclear envelope and the release of IL-12 but not IL-4 by vesicular transport. Thus, LIR7 is an activating receptor for eosinophils that elicited the release of cytotoxic granule proteins,de novolipid mediator generation, and cytokine release through vesicular transport.
Reactivity-based one-pot total synthesis of fucose GM
1
oligosaccharide: A sialylated antigenic epitope of small-cell lung cancer
Authors: Mong Tony Kwok-Kong , Lee Hing-Ken , Durón Sergio G. , Wong Chi-Huey
Published: February 2003

The total synthesis of the sialic acid-containing antigenic epitope fucose GM
1
(Fuc-GM
1
) by an improved reactivity-based one-pot synthetic strategy is reported. Based on a thioglycoside reactivity database, three saccharide building blocks, 3, 4, and 5, were designed and prepared to incorporate a descending order of reactivity toward thiophilic activation. Using the reactivity-based one-pot synthetic method, the fully protected Fuc-GM
1
glycoside 2 was furnished in a facile manner, which was globally deprotected to give the Fuc-GM
1
glycoside 1. In addition, using the promoter system 1-(benzensulfinyl)piperidine/trifluoromethanesulfonic anhydride, the product yield was improved and the reaction time was reduced in comparison with the
N
-iodosuccinimide/trifluoromethanesulfonic acid- and dimethyl (thiomethyl) sulfonium trifluoromethanesulfonate-promoted systems.
Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2
Authors: Leung K. C. , Doyle N. , Ballesteros M. , Sjogren K. , Watts C. K. W. , Low T. H. , Leong G. M. , Ross R. J. M. , Ho K. K. Y.
Published: February 2003

Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-α expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the β-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17β-estradiol (E
2
) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49–66% of control at 100 nM (
P
< 0.05). No reduction was seen when E
2
was added 1–2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E
2
suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E
2
inhibition. E
2
increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E
2
was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GH/JAK/STAT pathway, in which SOCS-2 plays a central mechanistic role.
Requirement of the calcineurin subunit gene
canB2
for indirect flight muscle formation in
Drosophila
Authors: Gajewski Kathleen , Wang Jianbo , Molkentin Jeffery D. , Chen Elizabeth H. , Olson Eric N. , Schulz Robert A.
Published: February 2003

Calcineurin is a calcium-activated protein phosphatase involved in multiple aspects of cardiac and skeletal muscle development and disease. Genes encoding calcineurin subunit proteins are highly conserved among animal species. Toward the goal of identifying new calcineurin-interacting loci that function in myogenic processes, we expressed an activated form of mouse calcineurin A in
Drosophila
and screened for suppressors of the phosphatase-induced lethality. Here, we demonstrate that a mutation in the
canB2
gene, which encodes a regulatory subunit of
Drosophila
calcineurin, can suppress a pupal developmental arrest phenotype to adult viability. As
canB2
is an essential gene and rare homozygous escapers are flightless, we further analyzed
canB2
expression and function in pupae and adults. The gene is expressed in the forming indirect flight muscles and central nervous system during pupal development. A
canA
gene is comparably expressed in these tissues. Consistent with the observed muscle expression,
canB2
mutants exhibit severe defects in the organization of their indirect flight muscles, a phenotype that is likely caused by muscle hypercontractility. Together, these findings demonstrate a vital role for the phosphatase in this specific facet of
Drosophila
myogenesis and show conserved fly and vertebrate calcineurin genes contribute prominently to fundamental processes of muscle formation and function.
GABAergic synapses made by a retinal dopaminergic neuron
Authors: Contini Massimo , Raviola Elio
Published: February 2003

In the retina, dopaminergic amacrine (interplexiform) cells establish multiple synapses on the perikarya of AII amacrines, the neurons that distribute rod signals to on- and off-cone bipolars. We used triple-label immunocytochemistry and confocal microscopy to identify the receptors contained within the postsynaptic active zone of these synapses in both mouse and rat retinas. We found that at the interface between the dendrites of the dopaminergic neurons and the AII amacrine cell perikarya clusters of postsynaptic γ-aminobutyric acid type A (GABA
A
) receptors are situated in register with aggregates of presynaptic organelles immunoreactive for GABA, the GABA vesicular transporter, and the vesicular monoamine transporter-2. D1 and D2/3 dopamine receptors, on the other hand, do not form clusters on the surface of the perikarya of AII amacrine cells. We suggest that the synapses between retinal dopaminergic neurons and AII amacrine cells are GABAergic and that both GABA and dopamine are released by the presynaptic endings. GABA acts on the ionotropic receptors clustered at the postsynaptic active zone, whereas dopamine diffuses to more distant, slower-acting metabotropic receptors.
Engineering the metabolism of
Escherichia coli
W3110 for the conversion of sugar to redox-neutral and oxidized products: Homoacetate production
Authors: Causey T. B. , Zhou S. , Shanmugam K. T. , Ingram L. O.
Published: February 2003

Microbial processes for commodity chemicals have focused on reduced products and anaerobic conditions where substrate loss to cell mass and CO
2
are minimal and product yields are high. To facilitate expansion into more oxidized chemicals,
Escherichia coli
W3110 was genetically engineered for acetate production by using an approach that combines attributes of fermentative and oxidative metabolism (rapid growth, external electron acceptor) into a single biocatalyst. The resulting strain (TC36) converted 333 mM glucose into 572 mM acetate, a product of equivalent oxidation state, in 18 h. With excess glucose, a maximum of 878 mM acetate was produced. Strain TC36 was constructed by sequentially assembling deletions that inactivated oxidative phosphorylation (Δ
atpFH
), disrupted the cyclic function of the tricarboxylic acid pathway (Δ
sucA
), and eliminated native fermentation pathways (Δ
focA-pflB
Δ
frdBC
Δ
ldhA
Δ
adhE
). These mutations minimized the loss of substrate carbon and the oxygen requirement for redox balance. Although TC36 produces only four ATPs per glucose, this strain grows well in mineral salts medium and has no auxotrophic requirement. Glycolytic flux in TC36 (0.3 μmol⋅min
−1
⋅mg
−1
protein) was twice that of the parent. Higher flux was attributed to a deletion of membrane-coupling subunits in (F
1
F
0
)H
+
-ATP synthase that inactivated ATP synthesis while retaining cytoplasmic F
1
-ATPase activity. The effectiveness of this deletion in stimulating flux provides further evidence for the importance of ATP supply and demand in the regulation of central metabolism. Derivatives of TC36 may prove useful for the commercial production of a variety of commodity chemicals.
The role of nuclear factor-κB essential modulator (NEMO) in B cell development and survival
Authors: Kim Shinyop , La Motte-Mohs Ross N. A. , Rudolph Dorothea , Zúñiga-Pflücker Juan Carlos , Mak Tak W.
Published: February 2003

The transcription factor nuclear factor-κB (NF-κB) is essential for immune and inflammatory responses. NF-κB essential modulator (NEMO) is a scaffolding component of the IκB kinase complex required for NF-κB activationin vitro. Because NF-κB activation is involved in B cell development and function, we set out to determine whether NEMO is required for these processes. NEMO(−/−) mice die very early during embryogenesis, and fetal livers from NEMO(−/−) embryos can not reconstitute either B or T lymphopoiesis in irradiated host mice. We therefore used NEMO(−/−) embryonic stem cells and the OP9in vitrodifferentiation system to demonstrate that NEMO is not required for B cell development but plays an important role in B cell survival.
Understanding neural system dynamics through task modulation and measurement of functional MRI amplitude, latency, and width
Authors: Bellgowan P. S. F. , Saad Z. S. , Bandettini P. A.
Published: February 2003

Estimates of hemodynamic amplitude, delay, and width were combined to investigate system dynamics involved in lexical decision making. Subjects performed a lexical decision task using word and nonword stimuli rotated 0°, 60°, or 120°. Averaged hemodynamic responses to repeated stimulation were fit to a Gamma-variate function convolved with a heavyside function of varying onset and duration to estimate each voxel's activation delay and width. Consistent with prolonged reaction times for the rotated stimuli and nonwords, the motor cortex showed delayed hemodynamic onset for both conditions. Language areas such as the lingual gyrus, middle temporal gyrus, fusiform gyrus, and precuneus all showed delayed hemodynamic onsets to rotated stimuli but not to nonword stimuli. The inferior frontal gyrus showed both increased onset latency for rotated stimuli and a wider hemodynamic response to nonwords, consistent with prolonged processing in this area during the lexical decision task. Phonological processing areas such as superior temporal and angular gyrus showed no delay or width difference for rotated stimuli. These results suggest that phonological routes but not semantic routes to the lexicon can proceed regardless of stimulus orientation. This study demonstrates the utility of estimating hemodynamic delay and width in addition to amplitude allowing for more quantitative measures of brain function such as mental chronometry.
Strikingly higher frequency in centenarians and twins of mtDNA mutation causing remodeling of replication origin in leukocytes
Authors: Zhang Jin , Asin-Cayuela Jordi , Fish Jennifer , Michikawa Yuichi , Bonafé Massimiliano , Olivieri Fabiola , Passarino Giuseppe , De Benedictis Giovanna , Franceschi Claudio , Attardi Giuseppe
Published: February 2003

The presence of a genetic component in longevity is well known. Here, the association of a mtDNA mutation with a prolonged life span in humans was investigated. Large-scale screening of the mtDNA main control region in leukocytes from subjects of an Italian population revealed a homoplasmic C150T transition near an origin of heavy mtDNA-strand synthesis in ≈17% of 52 subjects 99–106 years old, but, in contrast, in only 3.4% of 117 younger individuals (
P
= 0.0035). Evidence was obtained for the contribution of somatic events, under probable nuclear genetic control, to the striking selective accumulation of the mutation in centenarians. In another study, among leukocyte mtDNA samples from 20 monozygotic and 18 dizygotic twins, 60–75 years old, 30% (
P
= 0.0007) and 22% (
P
= 0.011), respectively, of the individuals involved exhibited the homoplasmic C150T mutation. In a different system, i.e., in five human fibroblast longitudinal studies, convincing evidence for the aging-related somatic expansion of the C150T mutation, up to homoplasmy, was obtained. Most significantly, 5′ end analysis of nascent heavy mtDNA strands consistently revealed a new replication origin at position 149, substituting for that at 151, only in C150T mutation-carrying samples of fibroblasts or immortalized lymphocytes. Considering the aging-related health risks that the centenarians have survived and the developmental risks of twin gestations, it is proposed that selection for a remodeled replication origin, inherited or somatically acquired, provides a survival advantage and underlies the observed high incidence of the C150T mutation in centenarians and twins.
Chromatin insulation by a transcriptional activator
Authors: Sutter Nathan B. , Scalzo David , Fiering Steven , Groudine Mark , Martin David I. K.
Published: February 2003

In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression.
A single-stranded promoter for RNA polymerase III
Authors: Schröder Oliver , Geiduschek E. Peter , Kassavetis George A.
Published: February 2003

Single strands of DNA serve, in rare instances, as promoters for transcription; duplex DNA promoters with individual strands that also have a promoter capacity/function have not been described. We show that the nontranscribed strand of the
Saccharomyces cerevisiae
U6 snRNA gene directs transcription initiation factor IIIB-requiring and accurately initiating transcription by RNA polymerase III. The nontranscribed strand promoter is much more extended than its duplex DNA counterpart, comprising the U6 gene TATA box, a downstream T
7
tract, and an upstream-lying segment. A requirement for placement of the 3′ end of the transcribed (template) strand within the confines of the transcription bubble is seen as indicating that the nontranscribed strand provides a scaffold for RNA polymerase recruitment but is deficient at a subsequent step of transcription initiation factor IIIB's direct involvement in promoter opening.
Molecular features of adult mouse small intestinal epithelial progenitors
Authors: Stappenbeck Thaddeus S. , Mills Jason C. , Gordon Jeffrey I.
Published: February 2003

The adult mouse small intestinal epithelium undergoes perpetual regeneration, fueled by a population of multipotential stem cells and oligopotential daughters located at the base of crypts of Lieberkühn. Although the morphologic features of small intestinal epithelial progenitors (SiEPs) are known, their molecular features are poorly defined. Previous impediments to purification and molecular characterization of SiEPs include lack of
ex vivo
clonigenic assays and the difficulty of physically retrieving them from their niche where they are interspersed between their numerous differentiated Paneth cell daughters. To overcome these obstacles, we used germ-free transgenic mice lacking Paneth cells to obtain a consolidated population of SiEPs with normal proliferative activity. These cells were harvested by laser capture microdissection. Functional genomics analysis identified 163 transcripts enriched in SiEPs compared with Paneth cell-dominated normal crypt base epithelium. The dataset was validated by (
i
) correlation with the organellar composition of SiEPs versus Paneth cells, (
ii
) similarities to databases generated from recent mouse hematopoietic and neural stem cell genome anatomy projects, and (
iii
) laser capture microdissection/real-time quantitative RT-PCR studies of progenitor cell-containing populations retrieved from the small intestines, colons, and stomachs of conventionally raised mice. The SiEP profile has prominent representation of genes involved in c-myc signaling and in the processing, localization, and translation of mRNAs. This dataset, together with our recent analysis of gene expression in the gastric stem cell niche, discloses a set of molecular features shared by adult mouse gut epithelial progenitors.