Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Phylogeographical footprints of the Strait of Gibraltar and Quaternary climatic fluctuations in the western Mediterranean: a case study with the greater white-toothed shrew, Crocidura russula (Mammalia: Soricidae).

We used mitochondrial cyt b sequences to investigate the phylogenetic relationships of Crocidura russula (sensu lato) populations across the Strait of Gibraltar, western Europe, Maghreb, and the Mediterranean and Atlantic islands. This revealed very low genetic divergence between European and Moroccan populations. The application of a molecular clock previously calibrated for shrews suggested that the separation of European from Moroccan lineages occurred less than 60 000 bp, which is at least 5 million years (Myr) after the reopening of the Strait of Gibraltar. This means that an overwater dispersal event was responsible for the observed phylogeographical structure. In contrast, genetic analyses revealed that Moroccan populations were highly distinct from Tunisian ones. According to the molecular clock, these populations separated about 2.2 million years ago (Ma), a time marked by sharp alternations of dry and humid climates in the Maghreb. The populations of the Mediterranean islands Ibiza, Pantelleria, and Sardinia were founded from Tunisian populations by overwater dispersal. In conclusion, overwater dispersal across the Strait of Gibraltar, probably assisted by humans, is possible for small terrestrial vertebrates. Moreover, as in Europe, Quaternary climatic fluctuations had a major effect on the phylogeographical structure of the Maghreb biota.

Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding.

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.

Native-like, long synthetic peptides as components of sub-unit vaccines: practical and theoretical considerations for their use in humans.

Vaccines have been used as a successful tool in medicine by way of controlling many major diseases. In spite of this, vaccines today represent only a handful of all infectious diseases. Therefore, there is a pressing demand for improvements of existing vaccines with particular reference to higher efficacy and undisputed safety profiles. To this effect, as an alternative to available vaccine technologies, there has been a drive to develop vaccine candidate polypeptides by chemical synthesis. In our laboratory, we have recently developed a technology to manufacture long synthetic peptides of up to 130 residues, which are correctly folded and biologically active. This paper discusses the advantages of the molecularly defined, long synthetic peptide approach in the context of vaccine design, development and use in human vaccination.

Reversible behavioural autistic-like regression: A manifestation of a special (new?) epileptic syndrome in a 28-month-old child. A 2-year longitudinal study .

A 28-month-old boy was referred for acute onset of abnormal head movements. History revealed an insidious progressive regression in behaviour and communication over several months. Head and shoulder 'spasms' with alteration of consciousness and on one occasion ictal laughter were seen. The electroencephalograph (EEG) showed repeated bursts of brief generalized polyspikes and spike-wave during the 'spasms', followed by flattening, a special pattern which never recurred after treatment. Review of family videos showed a single 'minor' identical seizure 6 months previously. Magnetic resonance imaging was normal. Clonazepam brought immediate cessation of seizures, normalization of the EEG and a parallel spectacular improvement in communication, mood and language. Follow-up over the next 10 months showed a new regression unaccompained by recognized seizures, although numerous seizures were discovered during the videotaped neuropsychological examination, when stereotyped subtle brief paroxysmal changes in posture and behaviour could be studied in slow motion and compared with the 'prototypical' initial ones. The EEG showed predominant rare left-sided fronto-temporal discharges. Clonazepam was changed to carbamazepin with marked improvement in behaviour, language and cognition which has been sustained up to the last control at 51 months. Videotaped home observations allowed the documentation of striking qualitative and quantitative variations in social interaction and play of autistic type in relation to the epileptic activity. We conclude that this child has a special characteristic epileptic syndrome with subtle motor and vegetative symptomatology associated with an insidious catastrophic 'autistic-like' regression which could be overlooked. The methods used to document such fluctuating epileptic behavioural manifestations are discussed.

Heterologous desensitization of the glucagon-like peptide-1 receptor by phorbol esters requires phosphorylation of the cytoplasmic tail at four different sites.

Glucagon-like peptide-1 stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor that activates the adenylyl cyclase pathway. We previously demonstrated that heterologous desensitization of the receptor by protein kinase C correlated with phosphorylation in a 33-amino acid-long segment of the receptor carboxyl-terminal cytoplasmic tail. Here, we determined that the in vivo sites of phosphorylation are four serine doublets present at positions 431/432, 441/442, 444/445, and 451/452. In vitro phosphorylation of fusion proteins containing mutant receptor C-tails, however, indicated that whereas serines at position 431/432 were good substrates for protein kinase C (PKC), serines 444/445 and 451/452 were poor substrates, and serines 441/442 were not substrates. In addition, serine 416 was phosphorylated on fusion protein but not in intact cells. This indicated that in vivo a different PKC isoform or a PKC-activated kinase may phosphorylate the receptor. The role of phosphorylation on receptor desensitization was assessed using receptor mutants expressed in COS cells or Chinese hamster lung fibroblasts. Mutation of any single serine doublet to alanines reduced the extent of phorbol 12-myristate 13-acetate-induced desensitization, whereas substitution of any combination of two serine doublets suppressed it. Our data thus show that the glucagon-like peptide-1 receptor can be phosphorylated in response to phorbol 12-myristate 13-acetate on four different sites within the cytoplasmic tail. Furthermore, phosphorylation of at least three sites was required for desensitization, although maximal desensitization was only achieved when all four sites were phosphorylated.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Genetic survey of "spinturnix sp" (Acari, Mesostigmata) and their bat hosts in Western Palearctic

SUMMARY : The coevolution between two intimately associated organisms, like host and parasite, is a widely investigated theme in evolutionary biology. Recently, the use of genetic data in the study of host-parasite systems evidences that the genetic information from some parasites can complement genetic data from their hosts and thus may help to better understand their host's evolutionary history. Phylogenetic and population genetic aspects of bat parasites have been poorly investigated. Spinturnicid mites are highly specialized ectoparasites, exclusively associated with bats and therefore represent an ideal model to extant our knowledge on bat and parasite biology and on their coevolutionary history. In this thesis, I developed several molecular markers (mitochondrial DNA) to compare the genetic patterns of Spinturnix mites with their bat hosts at different levels. The molecular co-phylogeny between Spinturnix sp. and their bat hosts suggests a partial cospeciation and the occurrence of failure to speciate events and multiple host switches. Thus, Spinturnix mites do not exactly mirror the phylogenetic pattern of their hosts, despite their intimate association. Similar roosting habits of the hosts seem to promote host switches between different species, as far as ecological conditions are favourable. The phylogeographic study of the Maghrebian bat M. punicus in the Mediterranean area confirms the presence of M. punicus in North Africa, Corsica and Sardinia and highlights that islands and mainland are genetically highly divergent. The comparison between the parasitic mite S. myoti and the Maghrebian bat suggests that the phylogeographic pattern of the mite is moulded by its host, with open water as main barrier for host and parasite dispersal. Moreover, the unique presence of a European S. myoti lineage on M. punicus from Corsica strongly suggests the former presence of mouse-eared bats (M. myotis and/or M. blythii) in Corsica. By highlighting the probable presence of a nowadays locally extinct host species, S. myoti may represent a good proxy for inferring complex evolutionary history of bat hosts. Finally, population genetic surveys of S. myoti and S. bechsteinii suggest that these mites benefit from close contacts between individuals during the mating season and/or hibernation to disperse among remote colonies. The contrasted genetic patterns of these two distinct bat-mite systems evidence that bat social structure is a determinant factor of the genetic structure of mite populations. Altogether, this PhD thesis demonstrates the usefulness of parasites to gather information about their bat hosts. In addition, my results illustrate how different ecological and biological characteristics of bat species allow the emergence of a surprising diversity in the genetic patterns of the parasites, which may contribute to the diversification and speciation of parasites. RESUME : La co-évolution entre deux organismes intimement liés, comme un parasite et son hôte, fait partie des questions largement étudiées en biologie évolutive. Récemment, l'utilisation de données génétique dans l'étude des interactions hôte-parasite a montré que l'information génétique de certains parasites peut compléter les données génétiques de l'hôte et ainsi peut éclairer l'histoire évolutive de leur hôte. Très peu études ont étudié les interactions entre les chauves-souris et leurs parasites d'un point de vue moléculaire. Les acariens du genre Spinturnix sont des ectoparasites très spécialisés exclusivement associés aux chauves-souris. Ils représentent donc un model idéal pour élargir nos connaissances tant sur l'écologie des parasites de chauves-souris que sur leur coévolution. Durant cette thèse, plusieurs marqueurs moléculaires (ADN mitochondrial) ont été développés pour ainsi comparer la distribution de la variation génétique des parasites du genre Spinturnix avec celle de leurs hôtes, et ceci à différents niveaux. Tout d'abord, la co-phylogénie moléculaire entre les espèces de Spinturnix et les leurs hôtes révèle une co-spéciation partielle ainsi que la présence d'événement de non spéciation et de transferts horizontaux. Ces parasites ne reflètent donc pas entièrement l'histoire évolutive de leurs hôtes, malgré leurs intimes associations. La cohabitation de plusieurs espèces de chauves-souris dans un même gîte permet aux parasites un transfert entre différentes espèces, atténuant ainsi leur degré de co-spéciation. Deuxièmement, l'étude phylogéographique du marin du Maghreb dans le bassin Méditerranéen confirme sa présence en Afrique du Nord, en Corse et en Sardaigne. La comparaison avec un de ses parasites S. myoti suggère que la répartition génétique de S. myoti est façonnée par celle de leurs hôtes, avec les étendues d'eau comme barrière principale tant à la dispersion de l'hôte que de son parasite. De plus, la présence unique d'une lignée européenne de ces parasites sur des marins du Maghreb de Corse suggère fortement la présence du grand ou petit marin en Corse dans le passé. En reflétant la présence potentielle à un endroit donné d'une espèce de chauve-souris actuellement disparue, S. myoti peut représenter une bonne alternative pour comprendre l'histoire évolutive complexe des chauves-souris. Finalement, l'étude des structures génétiques des populations des parasites S. myoti et S. bechsteinii suggère que les contacts corporels entre chauves-souris durant la saison de reproduction ou l'hibernation peuvent permettre la dispersion des parasites entre des colonies éloignées géographiquement. La différence de structure génétique entre ces deux associations particulières montre que la structure génétique des populations de parasites dépend fortement des traits d'histoire de vie de son hôte. Dans l'ensemble, cette thèse démontre l'importance des parasites pour amener des informations sur leurs hôtes, les chauves-souris. Elle illustre aussi comment les différences écologique et biologique des différentes espèces de chauves-souris peuvent amener une étonnante diversité de structure génétique au sein de populations de parasites, ce qui peut peut-être contribuer à la diversification et à la spéciation des parasites.

Autosomal-Recessive Posterior Microphthalmos Is Caused by Mutations in PRSS56, a Gene Encoding a Trypsin-Like Serine Protease.

Posterior microphthalmos (MCOP) is a rare isolated developmental anomaly of the eye characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal-recessive form (arMCOP) of the disease. Based on published linkage data, we refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in two large Faroese families. We detected three different mutations in PRSS56. Patients of the Faroese families were either homozygous for c.926G>C (p.Trp309Ser) or compound heterozygous for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in five patients with arMCOP from a consanguineous Tunisian family. In one patient with MCOP from the Faroe Islands and in another one from Turkey, no PRSS56 mutation was detected, suggesting nonallelic heterogeneity of the trait. Using RT-PCR, PRSS56 transcripts were detected in samples derived from the human adult retina, cornea, sclera, and optic nerve. The expression of the mouse ortholog could be first detected in the eye at E17 and was maintained into adulthood. The predicted PRSS56 protein is a 603 amino acid long secreted trypsin-like serine peptidase. The c.1066dupC is likely to result in a functional null allele, whereas the two point mutations predict the replacement of evolutionary conserved and functionally important residues. Molecular modeling of the p.Trp309Ser mutant suggests that both the affinity and reactivity of the enzyme toward in vivo protein substrates are likely to be substantially reduced.

Turning sunlight into stone: the oxalate-carbonate pathway in a tropical tree ecosystem.

An African oxalogenic tree, the iroko tree (Milicia excelsa), has the property to enhance carbonate precipitation in tropical oxisols, where such accumulations are not expected due to the acidic conditions in these types of soils. This uncommon process is linked to the oxalate-carbonate pathway, which increases soil pH through oxalate oxidation. In order to investigate the oxalate-carbonate pathway in the iroko system, fluxes of matter have been identified, described, and evaluated from field to microscopic scales. In the first centimeters of the soil profile, decaying of the organic matter allows the release of whewellite crystals, mainly due to the action of termites and saprophytic fungi. In addition, a concomitant flux of carbonate formed in wood tissues contributes to the carbonate flux and is identified as a direct consequence of wood feeding by termites. Nevertheless, calcite biomineralization of the tree is not a consequence of in situ oxalate consumption, but rather related to the oxalate oxidation inside the upper part of the soil. The consequence of this oxidation is the presence of carbonate ions in the soil solution pumped through the roots, leading to preferential mineralization of the roots and the trunk base. An ideal scenario for the iroko biomineralization and soil carbonate accumulation starts with oxalatization: as the iroko tree grows, the organic matter flux to the soil constitutes the litter, and an oxalate pool is formed on the forest ground. Then, wood rotting agents (mainly termites, saprophytic fungi, and bacteria) release significant amounts of oxalate crystals from decaying plant tissues. In addition, some of these agents are themselves producers of oxalate (e.g. fungi). Both processes contribute to a soil pool of "available" oxalate crystals. Oxalate consumption by oxalotrophic bacteria can then start. Carbonate and calcium ions present in the soil solution represent the end products of the oxalate-carbonate pathway. The solution is pumped through the roots, leading to carbonate precipitation. The main pools of carbon are clearly identified as the organic matter (the tree and its organic products), the oxalate crystals, and the various carbonate features. A functional model based on field observations and diagenetic investigations with δ13C signatures of the various compartments involved in the local carbon cycle is proposed. It suggests that the iroko ecosystem can act as a long-term carbon sink, as long as the calcium source is related to non-carbonate rocks. Consequently, this carbon sink, driven by the oxalate carbonate pathway around an iroko tree, constitutes a true carbon trapping ecosystem as defined by ecological theory.

Can the trabecular bone score be considered as a major clinical risk factor of osteoporotic fractures? A meta-like analysis.

To have an added value over BMD, a CRF of osteoporotic fracture must be predictable of the fracture, independent of BMD, reversible and quantifiable. Many major recognized CRF exist. Out of these factors many of them are indirect factor of bone quality. TBS predicts fracture independently of BMD as demonstrated from previous studies. The aim of the study is to verify if TBS can be considered as a major CRF of osteoporotic fracture. Existing validated datasets of Caucasian women were analyzed. These datasets stem from different studies performed by the authors of this report or provided to our group. However, the level of evidence of these studies will vary. Thus, the different datasets were weighted differently according to their design. This meta-like analysis involves more than 32000 women (≥50years) with 2000 osteoporotic fractures from two prospective studies (OFELY&MANITOBA) and 7 cross-sectional studies. Weighted relative risk (RR) for TBS was expressed for each decrease of one standard deviation as well as per tertile difference (TBS=1.300 and 1.200) and compared with those obtained for the major CRF included in FRAX®. Overall TBS RR obtained (adjusted for age) was 1.79 [95%CI-1.37-2.37]. For all women combined, RR for fracture for the lowest compared with the middle TBS tertile was 1.55[1.46-1.68] and for the lowest compared with the highest TBS tertile was 2.8[2.70-3.00]. TBS is comparable to most of the major CRF and thus could be used as one of them. Further studies have to be conducted to confirm these first findings.

Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1.

Glucagon-like peptide 1 (GLP-1) is a hormone derived from the preproglucagon molecule and is secreted by intestinal L cells. It is the most potent stimulator of glucose-induced insulin secretion and also suppresses in vivo acid secretion by gastric glands. A cDNA for the GLP-1 receptor was isolated by transient expression of a rat pancreatic islet cDNA library into COS cells; this was followed by binding of radiolabeled GLP-1 and screening by photographic emulsion autoradiography. The receptor transfected into COS cells binds GLP-1 with high affinity and is coupled to activation of adenylate cyclase. The receptor binds specifically GLP-1 and does not bind peptides of related structure and similar function, such as glucagon, gastric inhibitory peptide, vasoactive intestinal peptide, or secretin. The receptor is 463 amino acids long and contains seven transmembrane domains. Sequence homology is found only with the receptors for secretin, calcitonin, and parathyroid hormone, which form a newly characterized family of G-coupled receptors.