Multilocus definition of MHC haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis)

Cynomolgus macaques (Macaca fascicularis) are used widely in biomedical research, and the genetics of their MHC (Mhc-Mafa) has become the focus of considerable attention in recent years. The cohort of Indonesian pedigreed macaques that we present here was typed for Mafa-A, -B, and -DR, by sequencing, as described in earlier studies. Additionally, the DRB region of these animals was characterised by microsatellite analyses. In this study, full-length sequencing of Mafa-DPA/B and -DQA/B in these animals was performed. A total of 75 different alleles were observed; 22 of which have not previously been reported, plus 18 extended exon 2 alleles that were already known. Furthermore, two microsatellites, D6S2854 and D6S2859, were used to characterise the complex Mafa-A region. Sequencing and segregation analyses revealed that the length patterns of these microsatellites are unique for each Mafa-A haplotype. In this work, we present a pedigreed colony of approximately 120 cynomolgus macaques; all of which are typed for the most significant polymorphic MHC class I and class II markers. Offspring of these pedigreed animals are easily characterised for their MHC by microsatellite analyses on the Mafa-A and -DRB regions, which makes the cumbersome sequencing analyses redundant.     

Enhancement of Chemokine Function as an Immunomodulatory Strategy Employed by Human Herpesviruses

Herpes simplex virus (HSV) types 1 and 2 are highly prevalent human neurotropic pathogens that cause a variety of diseases, including lethal encephalitis. The relationship between HSV and the host immune system is one of the main determinants of the infection outcome. Chemokines play relevant roles in antiviral response and immunopathology, but the modulation of chemokine function by HSV is not well understood. We have addressed the modulation of chemokine function mediated by HSV. By using surface plasmon resonance and crosslinking assays we show that secreted glycoprotein G (SgG) from both HSV-1 and HSV-2 binds chemokines with high affinity. Chemokine binding activity was also observed in the supernatant of HSV-2 infected cells and in the plasma membrane of cells infected with HSV-1 wild type but not with a gG deficient HSV-1 mutant. Cell-binding and competition experiments indicate that the interaction takes place through the glycosaminoglycan-binding domain of the chemokine. The functional relevance of the interaction was determined both in vitro, by performing transwell assays, time-lapse microscopy, and signal transduction experiments; and in vivo, using the air pouch model of inflammation. Interestingly, and in contrast to what has been observed for previously described viral chemokine binding proteins, HSV SgGs do not inhibit chemokine function. On the contrary, HSV SgGs enhance chemotaxis both in vitro and in vivo through increasing directionality, potency and receptor signaling. This is the first report, to our knowledge, of a viral chemokine binding protein from a human pathogen that increases chemokine function and points towards a previously undescribed strategy of immune modulation mediated by viruses.     

Numbat: an interactive software tool for fitting Deltachi-tensors to molecular coordinates using pseudocontact shifts

Pseudocontact shift (PCS) effects induced by a paramagnetic lanthanide bound to a protein have become increasingly popular in NMR spectroscopy as they yield a complementary set of orientational and long-range structural restraints. PCS are a manifestation of the chi-tensor anisotropy, the Deltachi-tensor, which in turn can be determined from the PCS. Once the Deltachi-tensor has been determined, PCS become powerful long-range restraints for the study of protein structure and protein-ligand complexes. Here we present the newly developed package Numbat (New User-friendly Method Built for Automatic Deltachi-Tensor determination). With a Graphical User Interface (GUI) that allows a high degree of interactivity, Numbat is specifically designed for the computation of the complete set of Deltachi-tensor parameters (including shape, location and orientation with respect to the protein) from a set of experimentally measured PCS and the protein structure coordinates. Use of the program for Linux and Windows operating systems is illustrated by building a model of the complex between the E. coli DNA polymerase III subunits epsilon186 and theta using PCS.     

Prospects for lanthanides in structural biology by NMR

The advent of different lanthanide-binding reagents has made site-specific labelling of proteins with paramagnetic lanthanides a viable proposition. This brings many powerful techniques originally established and demonstrated for paramagnetic metalloproteins into the mainstream of structural biology. The promise is that, by exploiting the long-range effects of paramagnetism, lanthanide labelling will allow the study of larger proteins and protein-ligand complexes with greater ease and accuracy than hitherto possible. In particular, lanthanide-induced pseudocontact shifts (PCS) provide powerful restraints and 3D structure determination using PCS as the only source of experimental restraints will probably be possible with data obtained from samples with different lanthanide-tagging sites. Cell-free protein synthesis is positioned to play an important role in this strategy, as an inexpensive source of selectively labelled protein samples and for easy site-specific incorporation of unnatural lanthanide-binding amino acids.     

Inhibition of antibodies to CD3 surface antigen and phytohemagglutinin-mediated T cellular responses by inhibiting Ca2+/phospholipid-dependent protein kinase activity with the aid of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride

Ca2+/phospholipid-dependent kinase activity (C-kinase) plays an important second messenger role in T lymphocyte responses initiated by the cluster of differentiation (CD3) complex and presumably also lectinic receptors. During treatment with submitogenic or mitogenic amounts of phytohemagglutinin, as well as with anti-CD3 monoclonal antibody and 12-O-tetradecanoyl 13-phorbol acetate, the enzyme was intracellularly redistributed between the cytosol and the surface membrane. Submitogenic amounts of lectin and anti-CD3 were ineffective in inducing proliferation unless exogenous interleukin 2 (IL-2) was supplied, implying that even though IL-2 receptors were expressed, additional signals were required for IL-2 production. This would also indicate that there is a direct relationship between activation of C-kinase and expression of IL-2 receptors. The importance of C-kinase was further substantiated by the ability of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H7), a potent inhibitor of this enzyme, to interfere with IL-2 receptor expression and cellular [methyl-3H]thymidine uptake during primary activation. The drug concentration at which these cellular responses were inhibited by 50% was about the same as that which decreased c-kinase activity by 50% in vitro. H7 also prevented anti-CD3-induced translocation in intact cells. This effect may be related to competition with the phosphatidylserine binding site, which is important for membrane attachment. This drug apparently also interferes with the active center of the enzyme as demonstrated by its ability to inhibit Ca2+/phospholipid-independent phosphorylation of protamine sulfate. This additional mode of inhibition may be important in suppressing intact cell responses under circumstances during which the enzyme displacement to the membrane is nonphysiologic in nature, e.g., during treatment with 12-O-tetradecanoyl 13-phorbol acetate.     

The community succession of arthropods on a pig carcass in Lebanon: different taxonomic level approaches with faunistic notes

The diversity of arthropods is studied, at family and species level, on a pig carcass during a warm season in an open field in Badghan-Mount Lebanon. Ten orders of arthropods were found associated to the carcass. A total of 1168 specimens of adults Diptera and Coleoptera were collected. Among Diptera, the predator Chrysomya albiceps was dominant on the carcass during the active decay stage and Musca spp. were necrophagous. Within Coleoptera, Creophilus maxillosus (Linnaeus, 1758), Philonthus concinnus (Gravenhorst, 1802), Aleochara lata Gravenhorst, 1802, and Saprinus sp. were the predator taxa. Dermestes frischii Kugelann, 1792 and Necrobia rufipes (De Geer, 1775) were more present during the advanced decay, together with anthicids. In addition to a list of species visiting the carcass, we carried out a multivariate analysis of principal components to explore the relationships between the pattern of presences of 16 insect families and the four decomposition stages during the first 30 days of the carcass decomposition. The groupings of insects during the four decomposition stages show significant dissimilarities, suggesting that the different stages are characterized by different insect associations, as reported in many other previous studies.

Résumé. La succession des communautés d’arthropodes sur une carcasse de porc au Liban : approches à divers niveaux taxonomiques et notes faunistiques. La diversité des arthropodes est étudiée, au rang familial et spécifique, sur une carcasse de porc pendant la saison chaude en milieu naturel, sur le site de Badghan-Mount, au Liban. Dix ordres d’arthropodes ont été trouvés associés à la carcasse. Un total de 1168 spécimens adultes de Diptères et de Coléoptères a été collecté. Parmi les Diptères, le prédateur Chrysomya albiceps était dominant sur la carcasse au stade de décomposition active. Les Musca spp. étaient nécrophages. Parmi les Coléoptères, Creophilus maxillosus (Linnaeus, 1758), Philonthus concinnus (Gravenhorst, 1802), Aleochara lata Gravenhorst, 1802 et Saprinus sp. étaient prédateurs. Dermestes frischii Kugelann, 1792 et Necrobia rufipes (De Geer, 1775) et les Anthicidae étaient plus présents pendant la décomposition avancée. En complément à la liste des espèces visitant la carcasse, nous avons effectué une analyse multivariée en composantes principales pour explorer les relations entre le patron de présence de 16 familles d’insectes et les quatre stades de décomposition au cours des 30 premiers jours de la décomposition. Les groupements d’insectes montrent des dissimilarités significatives, ce qui suggère que les différents stades sont caractérisés par des associations différentes d’insectes, de façon analogue à ce qui a été rapporté dans plusieurs études antérieures.     

The wing base of the palaeodictyopteran genus Dunbaria Tillyard: Where are we now?

The structure of insect wing articulation is considered as reliable source of high level characters for phylogenetic analyses. However, the correct identification of homologous structures among the main groups of Pterygota is a hotly debated issue. Therefore, the reconstruction of the wing bases in Paleozoic extinct relatives is of great interest, but at the same time it should be treated with extreme caution due to distortions caused by taphonomic effects. The present study is focused on the wing base in Dunbaria (Spilapteridae). The articulation in Dunbaria quinquefasciata is mainly formed by a prominent upright axillary plate while the humeral plate is markedly reduced. Due to unique preservation of surface relief of the axillary plate, its composition shows a detailed pattern of three fused axillary sclerites and presumable position of the sclerite 3Ax. The obtained structures were compared among Spilapteridae and to other palaeodictyopterans Ostrava nigra (Homoiopteridae) and Namuroningxia elegans (Namuroningxiidae). The comparative study uncovered two patterns of 3Ax in Dunbaria and Namuroningxia, which correspond to their different suprafamilial classification. In contrast to previous studies these new results reveal the homologous structural elements in the wing base between Paleozoic Palaeodictyoptera and their extant relatives of Ephemeroptera, Odonata and Neoptera.