ORegAnno: an open access database and curation system for literature-derived promoters, transcription factor binding sites and regulatory variation.

MOTIVATION: Our understanding of gene regulation is currently limited by our ability to collectively synthesize and catalogue transcriptional regulatory elements stored in scientific literature. Over the past decade, this task has become increasingly challenging as the accrual of biologically validated regulatory sequences has accelerated. To meet this challenge, novel community-based approaches to regulatory element annotation are required. SUMMARY: Here, we present the Open Regulatory Annotation (ORegAnno) database as a dynamic collection of literature-curated regulatory regions, transcription factor binding sites and regulatory mutations (polymorphisms and haplotypes). ORegAnno has been designed to manage the submission, indexing and validation of new annotations from users worldwide. Submissions to ORegAnno are immediately cross-referenced to EnsEMBL, dbSNP, Entrez Gene, the NCBI Taxonomy database and PubMed, where appropriate. AVAILABILITY: ORegAnno is available directly through MySQL, Web services, and online at http://www.oreganno.org. All software is licensed under the Lesser GNU Public License (LGPL).

Embryonic enhancers in the dpp disk region regulate a second round of Dpp signaling from the dorsal ectoderm to the mesoderm that represses Zfh-1 expression in a subset of pericardial cells.

During germ band elongation, widespread decapentaplegic (dpp) expression in the dorsal ectoderm patterns the underlying mesoderm. These Dpp signals specify cardial and pericardial cell fates in the developing heart. At maximum germ band extension, dpp dorsal ectoderm expression becomes restricted to the dorsal-most or leading edge cells (LE). A second round of Dpp signaling then specifies cell shape changes in ectodermal cells leading to dorsal closure. Here we show that a third round of dpp dorsal ectoderm expression initiates during germ band retraction. This round of dpp expression is also restricted to LE cells but Dpp signaling specifies the repression of the transcription factor Zfh-1 in a subset of pericardial cells in the underlying mesoderm. Surprisingly, we found that cis-regulatory sequences that activate the third round of dpp dorsal ectoderm expression are found in the dpp disk region. We also show that the activation of this round of dpp expression is dependent upon prior Dpp signals, the signal transducer Medea, and possibly release from dTCF-mediated repression. Our results demonstrate that a second round of Dpp signaling from the dorsal ectoderm to the mesoderm is required to pattern the developing heart and that this round of dpp expression may be activated by combinatorial interactions between Dpp and Wingless.

Analysis of conserved noncoding DNA in Drosophila reveals similar constraints in intergenic and intronic sequences.

Comparative genomic approaches to gene and cis-regulatory prediction are based on the principle that differential DNA sequence conservation reflects variation in functional constraint. Using this principle, we analyze noncoding sequence conservation in Drosophila for 40 loci with known or suspected cis-regulatory function encompassing >100 kb of DNA. We estimate the fraction of noncoding DNA conserved in both intergenic and intronic regions and describe the length distribution of ungapped conserved noncoding blocks. On average, 22%-26% of noncoding sequences surveyed are conserved in Drosophila, with median block length approximately 19 bp. We show that point substitution in conserved noncoding blocks exhibits transition bias as well as lineage effects in base composition, and occurs more than an order of magnitude more frequently than insertion/deletion (indel) substitution. Overall, patterns of noncoding DNA structure and evolution differ remarkably little between intergenic and intronic conserved blocks, suggesting that the effects of transcription per se contribute minimally to the constraints operating on these sequences. The results of this study have implications for the development of alignment and prediction algorithms specific to noncoding DNA, as well as for models of cis-regulatory DNA sequence evolution.

The allometry of patch selection in ruminants.

An axiomatic feature of food consumption by animals is that intake rate and prey abundance are positively related. While this has been demonstrated rigorously for large herbivores, it is apparent from patch selection trials that grazers paradoxically tend to prefer short, sparse swards to tall, dense swards. Indeed, migratory herbivores often shift from areas of high to low sward biomass during the growing season. As nutritional quality is an inverse function of grass abundance, herbivores appear to sacrifice short-term intake for nutritional gains obtainable by eating sparse forage of higher quality. Explicit models of this trade-off suggest that individual ruminants maximize daily rates of energy gain by choosing immature swards of intermediate biomass. As body mass is related positively to both ruminant cropping rates and digestibility, there should be an allometric link between grass abundance and energy maximization, providing a tool for predicting patterns of herbivore habitat selection. We used previously published studies to develop a synthetic model of trade-offs between forage abundance and quality predicting that optimal sward biomass should scale allometrically with body size. The model predicts size-related variation in habitat selection observed in a guild of grazing ungulates in the Serengeti ecosystem.

Caribou movement as a correlated random walk.

Movement is a primary mechanism coupling animals to their environment, yet there exists little empirical analysis to test our theoretical knowledge of this basic process. We used correlated random walk (CRW) models and satellite telemetry to investigate long-distance movements of caribou, the most vagile, non-volant terrestrial vertebrate in the world. Individual paths of migratory and sedentary female caribou were quantified using measures of mean move length and angle, and net squared displacements at each successive move were compared to predictions from the models. Movements were modelled at two temporal scales. For paths recorded through one annual cycle, the CRW model overpredicted net displacement of caribou through time. For paths recorded over shorter intervals delineated by seasonal behavioural changes of caribou, there was excellent correspondence between model predictions and observations for most periods for both migratory and sedentary caribou. On the smallest temporal scale, a CRW model significantly overpredicted displacements of migratory caribou during 3 months following calving; this was also the case for sedentary caribou in late summer, and in late winter. In all cases of overprediction there was significant positive autocorrelation in turn direction, indicating that movements were more tortuous than expected. In one case of underprediction, significant negative autocorrelation of sequential turn direction was evident, indicating that migratory caribou moved in straightened paths during spring migration to calving grounds. Results are discussed in light of known migration patterns and possible limiting factors for caribou, and indicate the applicability of CRW models to animal movement at vast spatial and temporal scales, thus assisting in future development of more sophisticated models of population spread and redistribution for vertebrates.

Kinetics and cellular origin of cytokines in the central nervous system: insight into mechanisms of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 (H-2b) mice is characterized by early (day 12) acute paralysis, followed by a sustained chronic clinical course that gradually stabilizes. Extensive inflammation and demyelination coincide with clinical signs of disease. To identify the mechanisms of these processes, individual proinflammatory and anti-inflammatory cytokines and chemokines were studied. Sensitive single-cell assays were utilized to determine the cellular origin and kinetics of cytokine production in the CNS. Immunization with MOG35-55 peptide resulted in priming of both Th1 (lymphotoxin, IFN-gamma, and TNF-alpha) and Th2 (IL-4) cells in the spleen. However, only Th1 cells were apparent in the CNS. CD4 T cells that produced IFN-gamma or TNF-alpha were present in the CNS by day 7 after immunization with MOG35-55, peaked at day 20, and then waned. TNF-alpha was also produced in the CNS by Mac-1+ cells. On days 7 and 10 after immunization, the TNF-alpha-producing Mac1+ cells were predominantly microglia. By day 14, a switch occurred in that the Mac1+ TNF-alpha-producing cells had the phenotype of infiltrating macrophages. RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 chemokine mRNA were detected in the CNS by day 8 after immunization. The early presence of monocyte chemotactic protein 1 (MCP-1) in the CNS provides a mechanism for the recruitment of macrophages. These data implicate TNF-alpha production by a continuum of T cells, microglia, and macrophages at various times during the course of disease. The importance of Th1 cytokines is highlighted, with little evidence for a role of Th2 cytokines.

Lymphotoxin alpha3 induces chemokines and adhesion molecules: insight into the role of LT alpha in inflammation and lymphoid organ development.

Lymphotoxin (LT) plays an important role in inflammation and lymphoid organ development, though the mechanisms by which it promotes these processes are poorly understood. Toward this end, the biologic activities of a recently generated recombinant murine (m) LT alpha preparation were evaluated. This cytokine preparation was effective at inducing cytotoxicity of WEHI target cells with 50% maximal killing observed with 1.2 ng/ml. mLT alpha also induced the expression of inflammatory mediators in the murine endothelial cell line bEnd.3. rmLT alpha induced expression of the adhesion molecules VCAM, ICAM, E-selectin, and the mucosal addressin cellular adhesion molecule, MAdCAM-1. When mLT alpha, human (h) LT alpha, and mTNF-alpha were compared, mLT alpha was the most potent inducer of MAdCAM-1. None of these cytokines induced the peripheral node addressin, PNAd. mLT alpha also induced expression of the chemokines RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1). mRNA levels peaked 4 h following treatment with mLT alpha and declined through the 24-h treatment period. LT alpha also induced chemokine protein within 8 h of treatment, which increased through the 24-h treatment period. These data demonstrate that the proinflammatory effects of LT alpha3 may be mediated in part through the induction of adhesion molecule and chemokine expression. Further, LT alpha3 may promote development of lymphoid tissue through induction of chemokines and the mucosal addressin MAdCAM-1. These data confirm previous observations in transgenic and knockout mice that LT alpha3 in the absence of LT beta carries out unique biologic activities.

A critical role for lymphotoxin in experimental allergic encephalomyelitis.

The lymphotoxin (LT)/tumor necrosis factor (TNF) family has been implicated in the neurologic inflammatory diseases multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). To determine the role of individual family members in EAE, C57BL/6 mice, LT-alpha-deficient (LT-alpha-/- mice), or LT-beta-deficient (LT-beta-/- mice), and their wild-type (WT) littermates were immunized with rat myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. C57BL/6 and WT mice developed chronic, sustained paralytic disease with average maximum clinical scores of 3.5 and disease indices (a measure of day of onset and sustained disease scores) ranging from 367 to 663 with central nervous system (CNS) inflammation and demyelination. LT-alpha-/- mice were primed so that their splenic lymphocytes proliferated in response to MOG 35-55 and the mice produced anti-MOG antibody. However, LT-alpha-/- mice were quite resistant to EAE with low average clinical scores (<1), an average disease index of 61, and the negligible CNS inflammation and demyelination. WT T cells transferred EAE to LT-alpha-/- recipients. LT-beta-/- mice were susceptible to EAE, though less than WT, with an average maximum clinical score of 1.9 and disease index of 312. These data implicate T cell production of LT-alpha in MOG EAE and support a major role for LT-alpha3, a minor role for the LT-alpha/beta complex, and by inference, no role for TNF-alpha.

An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis.

Uncertainty regarding pathogenic mechanisms has been a major impediment to effective prevention and treatment for human neurologic diseases such as multiple sclerosis, tropical spastic paraparesis, and AIDS demyelinating disease. Here, we implicate lymphotoxin (LT) (tumor necrosis factor beta [TNF-beta]) and TNF-alpha in experimental allergic encephalomyelitis (EAE), a murine model of an autoimmune demyelinating disease. In this communication, we report that treatment of recipient mice with an antibody that neutralizes LT and TNF-alpha prevents transfer of clone-mediated EAE. LNC-8, a myelin basic protein-specific T cell line, produces high levels of LT and TNF-alpha after activation by concanavalin A, antibody to the CD-3 epsilon component of the T cell receptor, or myelin basic protein presented in the context of syngeneic spleen cells. LNC-8 cells transfer clinical signs of EAE. When LNC-8 recipient mice were also treated with TN3.19.12, a monoclonal antibody that neutralizes LT and TNF-alpha, the severity of the transferred EAE was reduced, while control antibodies did not alter the disease. The effect of anti-LT/TNF-alpha treatment was long lived and has been sustained for 5 mo. These findings suggest that LT and TNF-alpha and the T cells that produce them play an important role in EAE.

Subtypes of helper cells. Non-inflammatory type 1 helper T cells.

Class II MHC-restricted T cells recently have been characterized as being either type 1 (Th1) or type 2 (Th2) based on their ability to both secrete different lymphokines and perform different functions. Characterization of these subtypes to date have indicated that Th1 cells secrete IL-2, IFN-gamma, lymphotoxin, and IL-3, whereas Th2 cells secrete IL-4, IL-5, and IL-3. Functionally, Th1 cells mediated cytotoxicity and delayed-type hypersensitivity, and have been termed "inflammatory cells," whereas Th2 cells mediate helper function for Ig secretion and have been termed, "regulatory cells." We now present evidence that not all Th1 clones are inflammatory and capable of mediating cutaneous delayed-type hypersensitivity. We have generated a number of myelin basic protein-specific Th1 clones that do not mediate swelling when injected together with myelin basic protein directly into the footpads of syngeneic mice. These results suggest that Th1 cells can be further subdivided based on their ability to mediate delayed-type hypersensitivity, and that the Th1/Th2 characterization of Th cells may be insufficient to adequately characterize all functional subtypes of class II MHC-restricted T cells.