Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

Preliminary data on the geographical distribution of Drosophila species within morphoclimatic domains of Brazil. III. The cardini group

A reanalysis, based on museum specimens, of our previously published data on the geographical distribution of the species of Drosophila belonging to the cardini group in Brazil is presented and discussed. As previously recorded in several papers, including ours, the following four species were recognized: D. cardini, D. cardinoides, D. neocardini, and D. polymorpha. However, it was realized that most of the flies we have previously identified as Drosophila cardinoides belong in fact to Drosophila cardini. To facilitate the proper identification of these four near-sibling species, their holotypes were analyzed and their terminalia were described and illustrated. A key to the four species is also provided.

Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A.

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.

The fauna of phlebotomines (Diptera, Psychodidae) in different phytogeographic regions of the state of Maranhão, Brazil

Phlebotomine specimens were captured in domiciliary and forest environments in 47 municipalities between 1982 and 2005 with the aid of CDC light traps. A total of 91 species were found, of which four belonged to genus Brumptomyia and 87 to genus Lutzomyia, distributed among the following subgenera: Evandromyia (6), Lutzomyia (5), Micropygomyia (2), Nyssomyia (9), Pintomyia (2), Pressatia (3), Psathyromyia (6), Psychodopygus (14), Sciopemyia (4), Trichophoromyia (2), Viannamyia (2); species groups: Aragaoi (2), Baityi (1), Dreisbachi (1), Migonei (12), Oswaldoi (8), Pilosa (1), Saulensis (2), Verrucarum (4) and ungrouped (1). Species diversity was greatest in areas where there was dense evergreen seasonal forest (52 species), ombrophilous forest (31) and meridional cerrados (23) and lowest in areas with mixed forest (forest with babassu palms, cerrado and caatinga). The greatest similarity index was observed for restinga and open evergreen seasonal forest (J=0.48). Dense evergreen seasonal forest had greatest similarity with ombrophilous forest (J=0.38). The phlebotomine fauna was species rich and unevenly distributed in Maranhão, reflecting the phytogeographical complexity of the state, which is a result of the great variety of ecosystems and climate zones.

Protonation controls ASIC1a activity via coordinated movements in multiple domains.

Acid-sensing ion channels (ASICs) are neuronal Na(+)-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity.

Isolated integrin beta3 subunit cytoplasmic domains require membrane anchorage and the NPXY motif to recruit to adhesion complexes but do not discriminate between beta1- and beta3-positive complexes.

Integrin adhesion receptors consist of non-covalently linked alpha and beta subunits each of which contains a large extracellular domain, a single transmembrane domain and a short cytoplasmic tail. Engaged integrins recruit to focal structures globally termed adhesion complexes. The cytoplasmic domain of the beta subunit is essential for this clustering. beta1 and beta3 integrins can recruit at distinct cellular locations (i.e. fibrillar adhesions vs focal adhesions, respectively) but it is not clear whether individual beta subunit cytoplasmic and transmembrane domains are by themselves sufficient to drive orthotopic targeting to the cognate adhesion complex. To address this question, we expressed full-length beta3 transmembrane anchored cytoplasmic domains and truncated beta3 cytoplasmic domains as GFP-fusion constructs and monitored their localization in endothelial cells. Membrane-anchored full-length beta3 cytoplasmic domain and a beta3 mutant lacking the NXXY motif recruited to adhesion complexes, while beta3 mutants lacking the NPXY and NXXY motifs or the transmembrane domain did not. Replacing the natural beta subunit transmembrane domain with an unrelated (i.e. HLA-A2 alpha chain) transmembrane domain significantly reduced recruitment to adhesion complexes. Transmembrane anchored beta3 and cytoplasmic domain constructs, however, recruited without discrimination to beta1- and beta3-rich adhesions complexes. These findings demonstrate that membrane anchorage and the NPXY (but not the NXXY) motif are necessary for beta3 cytoplasmic domain recruitment to adhesion complexes and that the natural transmembrane domain actively contributes to this recruitment. The beta3 transmembrane and cytoplasmic domains alone are insufficient for orthotopic recruitment to cognate adhesion complexes.

Semantic domains and supersens tagging for domain-specific ontology learning

In this paper we propose a novel unsupervised approach to learning domain-specific ontologies from large open-domain text collections. The method is based on the joint exploitation of Semantic Domains and Super Sense Tagging for Information Retrieval tasks. Our approach is able to retrieve domain specific terms and concepts while associating them with a set of high level ontological types, named supersenses, providing flat ontologies characterized by very high accuracy and pertinence to the domain.

The Permian Pangea. Phytogeographic implications of new discoveries in Oman (Arabian Peninsula)

This paper focuses on new paleontological discoveries that have come to light while working on a Permian "paieobotanical" transect. from Oman to Central Morocco, In the Huqf area, the continental plant bearing "Gharif" Formation is bracketed by two welt dated marine Formations, providing new age constraint for the discovered "mixed" paleoHora, The newly named KubergarH;Jian'Murgabian "Gharif Paleoflora" is of outstanding paleogeographic .ignificance, Gondwanan, Cathaysian and Euramerian elements are found to be associated, The understanding of the kinematics of the Permian vegetal cover lead us to test the proposed Peri-Tethyan paleogeographical contours

Monte Carlo Study of the Growth of L12 ordered domains in fcc A3B binary alloys

A Monte Carlo study of the late time growth of L12-ordered domains in a fcc A3B binary alloy is presented. The energy of the alloy has been modeled by a nearest-neighbor interaction Ising Hamiltonian. The system exhibits a fourfold degenerated ground state and two kinds of interfaces separating ordered domains: flat and curved antiphase boundaries. Two different dynamics are used in the simulations: the standard atom-atom exchange mechanism and the more realistic vacancy-atom exchange mechanism. The results obtained by both methods are compared. In particular we study the time evolution of the excess energy, the structure factor and the mean distance between walls. In the case of atom-atom exchange mechanism anisotropic growth has been found: two characteristic lengths are needed in order to describe the evolution. Contrarily, with the vacancyatom exchange mechanism scaling with a single length holds. Results are contrasted with existing experiments in Cu3Au and theories for anisotropic growth.

Identification of Novel Craniofacial Regulatory Domains Located far Upstream of SOX9 and Disrupted in Pierre Robin Sequence.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.

The fibrinogen- and fibronectin-binding domains of Staphylococcus aureus fibronectin-binding protein A synergistically promote endothelial invasion and experimental endocarditis.

Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties--as well as elastin binding--and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by > or =10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.

The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host.

Plasmodium sporozoites make a remarkable journey from the mosquito midgut to the mammalian liver. The sporozoite's major surface protein, circumsporozoite protein (CSP), is a multifunctional protein required for sporozoite development and likely mediates several steps of this journey. In this study, we show that CSP has two conformational states, an adhesive conformation in which the C-terminal cell-adhesive domain is exposed and a nonadhesive conformation in which the N terminus masks this domain. We demonstrate that the cell-adhesive domain functions in sporozoite development and hepatocyte invasion. Between these two events, the sporozoite must travel from the mosquito midgut to the mammalian liver, and N-terminal masking of the cell-adhesive domain maintains the sporozoite in a migratory state. In the mammalian host, proteolytic cleavage of CSP regulates the switch to an adhesive conformation, and the highly conserved region I plays a critical role in this process. If the CSP domain architecture is altered such that the cell-adhesive domain is constitutively exposed, the majority of sporozoites do not reach their target organs, and in the mammalian host, they initiate a blood stage infection directly from the inoculation site. These data provide structure-function information relevant to malaria vaccine development.

Models that include supercoiling of topological domains reproduce several known features of interphase chromosomes.

Understanding the structure of interphase chromosomes is essential to elucidate regulatory mechanisms of gene expression. During recent years, high-throughput DNA sequencing expanded the power of chromosome conformation capture (3C) methods that provide information about reciprocal spatial proximity of chromosomal loci. Since 2012, it is known that entire chromatin in interphase chromosomes is organized into regions with strongly increased frequency of internal contacts. These regions, with the average size of ∼1 Mb, were named topological domains. More recent studies demonstrated presence of unconstrained supercoiling in interphase chromosomes. Using Brownian dynamics simulations, we show here that by including supercoiling into models of topological domains one can reproduce and thus provide possible explanations of several experimentally observed characteristics of interphase chromosomes, such as their complex contact maps.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells.

The Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA sub-domains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604-877) or A4(+16) (aa 432-559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.