Cutting edge: influence of the TCR V beta domain on the avidity of CD1d:alpha-galactosylceramide binding by invariant V alpha 14 NKT cells.

CD1d tetramers loaded with alpha-galactosylceramide (alpha-GalCer) bind selectively to mouse invariant Valpha14 (Valpha14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Valpha14-Jalpha18 chain in murine NKT cells, the associated beta-chain (typically expressing Vbeta8.2 or Vbeta7) appears not to influence tetramer binding. In this study, we describe novel alpha-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-beta chain on the avidity of CD1d:alpha-GalCer binding. A subset of Valpha14i NKT cells clearly discriminated alpha-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vbeta domain of the TCR-beta chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7.

Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

The InterPro Database, 2003 brings increased coverage and new features.

InterPro, an integrated documentation resource of protein families, domains and functional sites, was created in 1999 as a means of amalgamating the major protein signature databases into one comprehensive resource. PROSITE, Pfam, PRINTS, ProDom, SMART and TIGRFAMs have been manually integrated and curated and are available in InterPro for text- and sequence-based searching. The results are provided in a single format that rationalises the results that would be obtained by searching the member databases individually. The latest release of InterPro contains 5629 entries describing 4280 families, 1239 domains, 95 repeats and 15 post-translational modifications. Currently, the combined signatures in InterPro cover more than 74% of all proteins in SWISS-PROT and TrEMBL, an increase of nearly 15% since the inception of InterPro. New features of the database include improved searching capabilities and enhanced graphical user interfaces for visualisation of the data. The database is available via a webserver (http://www.ebi.ac.uk/interpro) and anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro).

Pressing the right buttons: signaling in lymphangiogenesis.

Lymphatic vasculature is increasingly recognized as an important factor both in the regulation of normal tissue homeostasis and immune response and in many diseases, such as inflammation, cancer, obesity, and hypertension. In the last few years, in addition to the central role of vascular endothelial growth factor (VEGF)-C/VEGF receptor-3 signaling in lymphangiogenesis, significant new insights were obtained about Notch, transforming growth factor ^6;/bone morphogenetic protein, Ras, mitogen-activated protein kinase, phosphatidylinositol 3 kinase, and Ca(2+)/calcineurin signaling pathways in the control of growth and remodeling of lymphatic vessels. An emerging picture of lymphangiogenic signaling is complex and in many ways distinct from the regulation of angiogenesis. This complexity provides new challenges, but also new opportunities for selective therapeutic targeting of lymphatic vasculature.

E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases.

E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.

New insights into the long-term evolutionary history of the avian MHC

SummaryGene duplication and neofunctidnalization are important processes in the evolution of phenotypic complexity. They account for important evolutionary novelties that confer ecological adaptation, such as the major histocompatibility complex (MHC), a multigene family with a central role in vertebrates' adaptive immune system. Multigene families, which evolved in large part through duplication, represent promising systems to study the still strongly depbated relative roles of neutral and adaptive processes in the evolution of phenotypic complexity. Detailed knowledge on ecological function and a well-characterized evolutionary history place the mammals' MHC amongst ideal study systems. However mammalian MHCs usually encompass several million base pairs and hold a large number of functional and non-functional duplicate genes, which makes their study complex. Avian MHCs on the other hand are usually way more compact, but the reconstruction of. their evolutionary history has proven notoriously difficult. However, no focused attempt has been undertaken so far to study the avian MHC evolutionary history in a broad phylogenetic context and using adequate gene regions.In the present PhD, we were able to make important contributions to the understanding of the long-term evolution of the avian MHC class II Β (MHCI1B). First, we isolated and characterized MHCIIB genes in barn owl (Tyto alba?, Strigiformes, Tytonidae), a species from an avian lineage in which MHC has not been studied so far. Our results revealed that with only two functional MHCIIB genes the MHC organization of barn owl may be similar to the 'minimal essential' MHC of chicken (Gallus gallus), indicating that simple MHC organization may be ancestral to birds. Taking advantage of the sequence information from barn owl, we studied the evolution of MHCIIB genes in 13 additional species of 'typical' owls (Strigiformes, Strigidae). Phylogenetic analyses revealed that according to their function, in owls the peptide-binding region (PBR) encoding exon 2 and the non-PBR encoding exon 3 evolve by different patterns. Exon 2 exhibited an evolutionary history of positive selection and recombination, while exon 3 traced duplication history and revealed two paralogs evolving divergently from each other in owls, and in a shorebird, the great snipe {Gallinago media). The results from exon 3 were the first ever from birds to demonstrate gene orthology in species that diverged tens of millions of years ago, and strongly questioned whether the taxa studied before provided an adequate picture of avian MHC evolution. In a follow-up study, we aimed at explaining a striking pattern revealed by phylogenetic trees analyzing the owl sequences along with MHCIIB sequences from other birds: One owl paralog (termed DAB1) grouped with sequences of passerines and falcons, while the other (DAB2) grouped with wildfowl, penguins and birds of prey. This could be explained by either a duplication event preceding the evolution of these bird orders, or by convergent evolution of similar sequences in a number of orders. With extensive phylogenetic analyses we were able to show, that indeed a duplication event preceeded the major avian radiation -100 my ago, and that following this duplication, the paralogs evolved under positive selection. Furthermore, we showed that the divergently evolving amino acid residues in the MHCIIB-encoded ^6;-chain potentially interact with the MHCI I α-chain, and that molecular coevolution of the interacting residues may have been involved in the divergent evolution of the MHCIIB paralogs.The findings of this PhD are of particular interest to the understanding of the evolutionary history of the avian MHC and, by providing essential information on long-term gene history in the avian MHC, open promising perspectives for advances in the understanding of the evolution of multigene families in general, and for avian MHC organization in particular. Amongst others I discuss the importance of including protein structure in the phylogenetic study of multigene families, and the roles of ecological versus molecular selection pressures. I conclude by providing a population genomic perspective on avian MHC, which may serve as a basis for future research to investigate the relative roles of neutral processes involving effective population size effects and of adaptation in the evolution of avian MHC diversity and organization.RésuméLa duplication de gènes et leur néo-fonctionnalisation sont des processus importants dans l'évolution de la complexité phénotypique. Ils sont impliqués dans l'apparition d'importantes nouveautés évolutives favorisant l'adaptation écologique, comme c'est le cas pour le complexe majeur d'histocompatibilité 6; codée par le CMHIIB interagissent potentiellement avec la chaîne α codée par le CMHII, et que la coévolution moléculaire de ces résidus pourrait être à la base de l'évolution divergente des paralogues CMHIIB.Dans cette thèse sont discutés entre autres l'importance d'inclure la structure des protéines dans l'étude phylogénétique des familles multigéniques, ainsi que les rôles respectifs des pressions sélectives écologique et moléculaire. La conclusion comporte une perspective sur la génomique des populations du CMH aviaire, qui pourrait servir de base pour de futures recherches sur les rôles relatifs joués par Îes processus neutres comprenant les effets de la taille efficace des populations et l'adaptation dans l'évolution de la diversité et l'organisation du MHC aviaire.

Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists.

The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, AF-1 in the N-terminal part of the receptor and AF-2 in its ligand-binding domain. AF-2 activity is dependent upon a putative amphipathic alpha-helix between residues 538 and 552 in the mouse ER. Point mutagenesis of conserved hydrophobic residues within this region reduces estrogen-dependent transcriptional activation without affecting hormone and DNA binding significantly. Here we show that these mutations dramatically alter the pharmacology of estrogen antagonists. Both tamoxifen and ICI 164,384 behave as strong agonists in HeLa cells expressing the ER mutants. In contrast to the wild-type ER, the mutant receptors maintain nuclear localization and DNA-binding activity after ICI 164,384 treatment. Structural alterations in AF-2 caused by gene mutations such as those described herein or by estrogen-independent signaling pathways may account for the insensitivity of some breast cancers to tamoxifen treatment.

Binding of d-methadone, l-methadone, and dl-methadone to proteins in plasma of healthy volunteers: role of the variants of alpha 1-acid glycoprotein.

The plasma concentrations of alpha 1-acid glycoprotein (AAG), albumin, triglycerides, cholesterol, and total proteins, as well as the plasma binding of racemic, d-methadone, and l-methadone were measured in 45 healthy subjects. The AAG phenotypes and the concentrations of AAG variants were also determined. The measured free fractions for racemic, d-methadone, and l-methadone were, respectively, 12.7% +/- 3.3%, 10.0% +/- 2.9%, and 14.2% +/- 3.2% (mean +/- SD). A significant correlation was obtained between the binding ratio (B/F) for dl-methadone and the total AAG concentration (r = 0.724; p less than 0.001). A multiple stepwise regression analysis showed that AAG was the main explanatory variable for the binding of the racemate. When concentrations of AAG variants were considered, a significant correlation was obtained between the binding ratio of dl-methadone and orosomucoid2 A concentration (r = 0.715; p less than 0.001), a weak correlation between dl-methadone and orosomucoid1 S concentration (r = 0.494; p less than 0.001), and no correlation between dl-methadone and orosomucoid1 F1 concentration (r = 0.049; not significant). Similar findings were obtained with the enantiomers. This study shows the importance of considering not only total AAG but also concentrations of AAG variants when measuring the binding of methadone and possibly of other drugs in plasma.

The vacuolar transporter chaperone (VTC) complex is required for microautophagy.

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes.

Combinatorial effects of splice variants modulate function of Aiolos

The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Acute imaging does not improve ASTRAL score's accuracy despite having a prognostic value.

BACKGROUND: The ASTRAL score was recently shown to reliably predict three-month functional outcome in patients with acute ischemic stroke. AIM: The study aims to investigate whether information from multimodal imaging increases ASTRAL score's accuracy. METHODS: All patients registered in the ASTRAL registry until March 2011 were included. In multivariate logistic-regression analyses, we added covariates derived from parenchymal, vascular, and perfusion imaging to the 6-parameter model of the ASTRAL score. If a specific imaging covariate remained an independent predictor of three-month modified Rankin score > 2, the area-under-the-curve (AUC) of this new model was calculated and compared with ASTRAL score's AUC. We also performed similar logistic regression analyses in arbitrarily chosen patient subgroups. RESULTS: When added to the ASTRAL score, the following covariates on admission computed tomography/magnetic resonance imaging-based multimodal imaging were not significant predictors of outcome: any stroke-related acute lesion, any nonstroke-related lesions, chronic/subacute stroke, leukoaraiosis, significant arterial pathology in ischemic territory on computed tomography angiography/magnetic resonance angiography/Doppler, significant intracranial arterial pathology in ischemic territory, and focal hypoperfusion on perfusion-computed tomography. The Alberta Stroke Program Early CT score on plain imaging and any significant extracranial arterial pathology on computed tomography angiography/magnetic resonance angiography/Doppler were independent predictors of outcome (odds ratio: 0·93, 95% CI: 0·87-0·99 and odds ratio: 1·49, 95% CI: 1·08-2·05, respectively) but did not increase ASTRAL score's AUC (0·849 vs. 0·850, and 0·8563 vs. 0·8564, respectively). In exploratory analyses in subgroups of different prognosis, age or stroke severity, no covariate was found to increase ASTRAL score's AUC, either. CONCLUSIONS: The addition of information derived from multimodal imaging does not increase ASTRAL score's accuracy to predict functional outcome despite having an independent prognostic value. More selected radiological parameters applied in specific subgroups of stroke patients may add prognostic value of multimodal imaging.

Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids.

5

Retinoid X Receptors (RXR) were initially identified as nuclear receptors binding with stereo-selectivity the vitamin A derivative 9-cis retinoic acid, although the relevance of this molecule as endogenous activator of RXRs is still elusive. Importantly, within the nuclear receptor superfamily, RXRs occupy a peculiar place, as they are obligatory partners for a number of other nuclear receptors, thus integrating the corresponding signaling pathways. In this chapter, we describe the structural features allowing RXR to form homo- and heterodimers, and the functional consequences of this unique ability. Furthermore, we discuss the importance of studying RXR activity at a genome-wide level in order to comprehensively address the biological implications of their action that is fundamental to understand to what extent RXRs could be exploited as new therapeutic targets.