Distinct conformations of Ly49 natural killer cell receptors mediate MHC class I recognition in trans and cis.

Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors and explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.

Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines.

The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA- or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.

Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

Genetics of colouration in birds.

Establishing the links between phenotype and genotype is of great importance for resolving key questions about the evolution, maintenance and adaptive function of phenotypic variation. Bird colouration is one of the most studied systems to investigate the role of natural and sexual selection in the evolution of phenotypic diversity. Given the recent advances in molecular tools that allow discovering genetic polymorphisms and measuring gene and protein expression levels, it is timely to review the literature on the genetics of bird colouration. The present study shows that melanin-based colour phenotypes are often associated with mutations at melanogenic genes. Differences in melanin-based colouration are caused by switches of eumelanin to pheomelanin production or by changes in feather keratin structure, melanoblast migration and differentiation, as well as melanosome structure. Similar associations with other types of colourations are difficult to establish, because our knowledge about the molecular genetics of carotenoid-based and structural colouration is quasi inexistent. This discrepancy stems from the fact that only melanin-based colouration shows pronounced heritability estimates, i.e. the resemblance between related individuals is usually mainly explained by genetic factors. In contrast, the expression of carotenoid-based colouration is phenotypically plastic with a high sensitivity to variation in environmental conditions. It therefore appears that melanin-based colour traits are prime systems to understand the genetic basis of phenotypic variation. In this context, birds have a great potential to bring us to new frontiers where many exciting discoveries will be made on the genetics of phenotypic traits, such as colouration. In this context, a major goal of our review is to suggest a number of exciting future avenues.

CREB-2, a cellular CRE-dependent transcription repressor, functions in association with Tax as an activator of the human T-cell leukemia virus type 1 promoter.

The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent.

NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production?

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a multiprotein complex that activates caspase 1, leading to the processing and secretion of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and IL-18. The NLRP3 inflammasome is activated by a wide range of danger signals that derive not only from microorganisms but also from metabolic dysregulation. It is unclear how these highly varied stress signals can be detected by a single inflammasome. In this Opinion article, we review the different signalling pathways that have been proposed to engage the NLRP3 inflammasome and suggest a model in which one of the crucial elements for NLRP3 activation is the generation of reactive oxygen species (ROS).

Molecular genetics of charcot-marie-tooth disease: from genes to genomes.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders of the peripheral nervous system, mainly characterized by distal muscle weakness and atrophy leading to motor handicap. With an estimated prevalence of 1 in 2,500, this condition is one of the most commonly inherited neurological disorders. Mutations in more than 30 genes affecting glial and/or neuronal functions have been associated with different forms of CMT leading to a substantial improvement in diagnostics of the disease and in the understanding of implicated pathophysiological mechanisms. However, recent data from systematic genetic screening performed in large cohorts of CMT patients indicated that molecular diagnosis could be established only in ∼50-70% of them, suggesting that additional genes are involved in this disease. In addition to providing an overview of genetic and functional data concerning various CMT forms, this review focuses on recent data generated through the use of highly parallel genetic technologies (SNP chips, sequence capture and next-generation DNA sequencing) in CMT families, and the current and future impact of these technologies on gene discovery and diagnostics of CMTs.

Genetics of calcium homeostasis in humans: continuum between monogenic diseases and continuous phenotypes

Extracellular calcium participates in several key physiological functions, such as control of blood coagulation, bone calcification or muscle contraction. Calcium homeostasis in humans is regulated in part by genetic factors, as illustrated by rare monogenic diseases characterized by hypo or hypercalcaemia. Both serum calcium and urinary calcium excretion are heritable continuous traits in humans. Serum calcium levels are tightly regulated by two main hormonal systems, i.e. parathyroid hormone and vitamin D, which are themselves also influenced by genetic factors. Recent technological advances in molecular biology allow for the screening of the human genome at an unprecedented level of detail and using hypothesis-free approaches, such as genome-wide association studies (GWAS). GWAS identified novel loci for calcium-related phenotypes (i.e. serum calcium and 25-OH vitamin D) that shed new light on the biology of calcium in humans. The substantial overlap (i.e. CYP24A1, CASR, GATA3; CYP2R1) between genes involved in rare monogenic diseases and genes located within loci identified in GWAS suggests a genetic and phenotypic continuum between monogenic diseases of calcium homeostasis and slight disturbances of calcium homeostasis in the general population. Future studies using whole-exome and whole-genome sequencing will further advance our understanding of the genetic architecture of calcium homeostasis in humans. These findings will likely provide new insight into the complex mechanisms involved in calcium homeostasis and hopefully lead to novel preventive and therapeutic approaches. Keyword: calcium, monogenic, genome-wide association studies, genetics.

Selective inhibition of CTL activation by a dipalmitoyl-phospholipid that prevents the recruitment of signaling molecules to lipid rafts.

Antigen-specific T-cell activation implicates a redistribution of plasma membrane-bound molecules in lipid rafts, such as the coreceptors CD8 and CD4, the Src kinases Lek and Fyn, and the linker for activation of T cells (LAT), that results in the formation of signaling complexes. These molecules partition in lipid rafts because of palmitoylation of cytoplasmic, membrane proximal cysteines, which is essential for their functional integrity in T-cell activation. Here, we show that exogenous dipalmitoyl-phosphatidylethanolamine (DPPE), but not the related unsaturated dioleoyl-phosphatidylethanolamine (DOPE), partitions in lipid rafts. DPPE inhibits activation of CD8(+) T lymphocytes by sensitized syngeneic antigen-presenting cells or specific major histocompatibility complex (MHC) peptide tetramers, as indicated by esterase release and intracellular calcium mobilization. Cytotoxic, T lymphocyte (CTL)-target cell conjugate formation is not affected by DPPE, indicating that engagement of the T-cell receptor by its cognate ligand is intact in lipid-treated cells. In contrast to other agents known to block raft-dependent signaling, DPPE efficiently inhibits the MHC peptide-induced recruitment of palmitoylated signaling molecules to lipid rafts and CTL activation without affecting cell viability or lipid raft integrity.

Immunocytochemical and pharmacological characterization of metabotropic glutamate receptors of the vestibular end organs in the frog.

Using immunocytochemistry and multiunit recording of afferent activity of the whole vestibular nerve, we investigated the role of metabotropic glutamate receptors (mGluR) in the afferent neurotransmission in the frog semicircular canals (SCC). Group I (mGluR1alpha) and group II (mGluR2/3) mGluR immunoreactivities were distributed to the vestibular ganglion neurons, and this can be attributed to a postsynaptic locus of metabotropic regulation of rapid excitatory transmission. The effects of group I/II mGluR agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) on resting and chemically induced afferent activity were studied. ACPD (10-100 microM) enhanced the resting discharge frequency. MCPG (5-100 microM) led to a concentration-dependent decrease of both resting activity and ACPD-induced responses. If the discharge frequency had previously been restored by L-glutamate (L-Glu) in high-Mg2+ solution, ACPD elicited a transient increase in the firing rate in the afferent nerve suggesting that ACPD acts on postsynaptic receptors. The L-Glu agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA), were tested during application of ACPD. AMPA- and NMDA-induced responses were higher in the presence than absence of ACPD, implicating mGluR in the modulation of ionotropic glutamate receptors. These results indicate that activation of mGluR potentiates AMPA and NMDA responses through a postsynaptic interaction. We conclude that ACPD may exert modulating postsynaptic effects on vestibular afferents and that this process is activity-dependent.

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response.

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4+ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4+ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35-/- and IL-12p40-/-) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4+ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in purified CD4+ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-gamma and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-gamma mRNA in CD4+ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-gamma in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.

BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.

Injection of partially purified estrogen receptor protein from Xenopus liver nuclei into oocytes activates the silent vitellogenin locus.

Injection of extracts from Xenopus liver nuclei that are enriched 2000 times in estradiol receptor into Xenopus oocytes induces transcription of the silent vitellogenin locus, which is activated in liver by estradiol, but not of the albumin locus, which is active in liver but suppressed by high levels of estradiol. Transcription initiates within the 5'-end region of the gene we have studied and probably continues into the 3' third. The activation seems to be very efficient, but most of the primary transcripts are probably rapidly and inaccurately processed. New proteins are also made and secreted by the oocytes.

Altered Local Beta and Gamma Synchronization in Prefrontal Cortex of Mice with Redox Dysregulation or Maternal Immune Activation

Background: A developmental dysregulation of glutathione (GSH) synthesis leading to oxidative stress, when combined with environmental risk factors (viral infections) generating reactive oxygen species, can play a critical role in inducing schizophrenia phenotypes. GSH deficit induces morphological, physiological and behavioral anomalies analogous to those reported in schizophrenic patients, including disrupted parvalbumine (PV) inhibitory interneuron's integrity and neuronal synchrony (β/γ-oscillations). Methods: We assessed PV immunoreactivity (PV-IR) and local synchronization in prefrontal cortex of two mouse models: (1) mice with a genetic deficit in GSH (GCLM-/-) and (2) mice with prenatal immune activation at embryonic day17 (PolyI:C). Results: Adults from both mice models display reduced PV-IR in prefrontal cortex. In anterior cingulate (ACC) of GCLM-/-, appearance and maturation of PVI are delayed and worsened with peribubertal stress but not in adult one. This effect is reversed by treatment with the GSH precursor N-acetyl-cysteine. The power of beta and gamma oscillations are decreased in ACC of GCLM-/- while they increased in prelimbic cortex of PolyI:C mice. Conclusions: Despite reduced PV-IR in both models, alteration of the synchronization was different, indicating that the structural/functional disruption of the cortical circuitry was partly different in both models. Novel therapeutic strategies are proposed, based on interference with oxidative stress and inflammatory processes.

Genetics of cadmium hyperaccumulation variance in Thlaspi caerulescens natural populations from Switzerland

SUMMARY Heavy metal presence in the environment is a serious concern since some of them can be toxic to plants, animals and humans once accumulated along the food chain. Cadmium (Cd) is one of the most toxic heavy metal. It is naturally present in soils at various levels and its concentration can be increased by human activities. Several plants however have naturally developed strategies allowing them to grow on heavy metal enriched soils. One of them consists in the accumulation and sequestration of heavy metals in the above-ground biomass. Some plants present in addition an extreme strategy by which they accumulate a limited number of heavy metals in their shoots in amounts 100 times superior to those expected for a non-accumulating plant in the same conditions. Understanding the genetic basis of the hyperaccumulation trait - particularly for Cd - remains an important challenge which may lead to biotechnological applications in the soil phytoremediation. In this thesis, Thlaspi caerulescens J. & C. Presl (Brassicaceae) was used as a model plant to study the Cd hyperaccumulation trait, owing to its physiological and genetic characteristics. Twenty-four wild populations were sampled in different regions of Switzerland. They were characterized for environmental and soil parameters as well as intrinsic characteristics of plants (i.e. metal concentrations in shoots). They were as well genetically characterized by AFLPs, plastid DNA polymorphism and genes markers (CAPS and microsatellites) mainly developed in this thesis. Some of the investigated genes were putatively linked to the Cd hyperaccumulation trait. Since the study of the Cd hyperaccumulation in the field is important as it allows the identification of patterns of selection, the present work offered a methodology to define the Cd hyperaccumulation capacity of populations from different habitats permitting thus their comparison in the field. We showed that Cd, Zn, Fe and Cu accumulations were linked and that populations with higher Cd hyperaccumulation capacity had higher shoot and reproductive fitness. Using our genetic data, statistical methods (Beaumont & Nichols's procedure, partial Mantel tests) were applied to identify genomic signatures of natural selection related to the Cd hyperaccumulation capacity. A significant genetic difference between populations related to their Cd hyperaccumulation capacity was revealed based on somè specific markers (AFLP and candidate genes). Polymorphism at the gene encoding IRTl (Iron-transporter also participating to the transport of Zn) was suggested as explaining part of the variation in Cd hyperaccumulation capacity of populations supporting previous physiological investigations. RÉSUMÉ La présence de métaux lourds dans l'environnement est un phénomène préoccupant. En effet, certains métaux lourds - comme le cadmium (Cd) -sont toxiques pour les plantes, les animaux et enfin, accumulés le long de la chaîne alimentaire, pour les hommes. Le Cd est naturellement présent dans le sol et sa concentration peut être accrue par différentes activités humaines. Certaines plantes ont cependant développé des stratégies leur permettant de pousser sur des sols contaminés en métaux lourds. Parmi elles, certaines accumulent et séquestrent les métaux lourds dans leurs parties aériennes. D`autres présentent une stratégie encore plus extrême. Elles accumulent un nombre limité de métaux lourds en quantités 100 fois supérieures à celles attendues pour des espèces non-accumulatrices sous de mêmes conditions. La compréhension des bases génétiques de l'hyperaccumulation -particulièrement celle du Cd - représente un défi important avec des applications concrètes en biotechnologies, tout particulièrement dans le but appliqué de la phytoremediation des sols contaminés. Dans cette thèse, Thlaspi caerulescens J. & C. Presl (Brassicaceae) a été utilisé comme modèle pour l'étude de l'hyperaccumulation du Cd de par ses caractéristiques physiologiques et génétiques. Vingt-quatre populations naturelles ont été échantillonnées en Suisse et pour chacune d'elles les paramètres environnementaux, pédologique et les caractéristiques intrinsèques aux plantes (concentrations en métaux lourds) ont été déterminés. Les populations ont été caractérisées génétiquement par des AFLP, des marqueurs chloroplastiques et des marqueurs de gènes spécifiques, particulièrement ceux potentiellement liés à l'hyperaccumulation du Cd (CAPS et microsatellites). La plupart ont été développés au cours de cette thèse. L'étude de l'hyperaccumulation du Cd en conditions naturelles est importante car elle permet d'identifier la marque, éventuelle de sélection naturelle. Ce travail offre ainsi une méthodologie pour définir et comparer la capacité des populations à hyperaccumuler le Cd dans différents habitats. Nous avons montré que les accumulations du Cd, Zn, Fe et Cu sont liées et que les populations ayant une grande capacité d'hyperaccumuler le Cd ont également une meilleure fitness végétative et reproductive. Des méthodes statistiques (l'approche de Beaumont & Nichols, tests de Martel partiels) ont été utilisées sur les données génétiques pour identifier la signature génomique de la sélection naturelle liée à la capacité d'hyperaccumuler le Cd. Une différenciation génétique des populations liée à leur capacité d'hyperaccumuler le Cd a été mise en évidence sur certains marqueurs spécifiques. En accord avec les études physiologiques connues, le polymorphisme au gène codant IRT1 (un transporteur de Fe impliqué dans le transport du Zn) pourrait expliquer une partie de la variance de la capacité des populations à hyperaccumuler le Cd.