ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Meningococcal disease in a kidney transplant recipient with mannose-binding lectin (MBL) deficiency

Background: There is an increasing amount of data associating MBL deficiency with a higher susceptibility to meningococca[ disease. In addition, meningococca[ disease has been reported in patients with various immunosuppressive conditions. However, to our knowledge, only three cases of meningococca[ disease have been reported in solid organ recipients (SOT). Methods & Results: A 32 year-old male patient underwent cadaveric kidney transplantation for endstage renal disease of unknown origin. On day 71 post-transplantation he developed fever (39.6°C), shaking chilis, and tachycardia without hypotension. At this time, immunosuppression consisted of tacro[imus, prednisone 10mg daily and mycopheno[ ate mofeti[ 2 g daily. Physical examination on admission was normal, except for two small petechia[ lesions on the forearm. No meningeal signs were present. Three sets of blood cultures grew Neisseria meningitidis group C susceptible to ceftriaxone (MIC=0.003mg/[). Antibiotic therapy consisted in intravenous ceftriaxone 2 g per day for a total duration of 7 days. Serum immunog[obu[in levels, C3, C4 and CHS0 were normal However, using a method to screen for the functional activity of a[[ three pathways of complement (Wies[ab, Lund, Sweden), no activation via the MBL pathway could be detected (0%). A subsequent quantification of MBL pathway components revealed normal levels of MASP 2 but undetectab[e amounts of MBL (below 10 ng/m[, normal range: >500 ng/m[). Conclusion: Since the exact incidence and the possible relationship between meningococca[ disease and organ transplantation is not we[[ understood, we strongly encourage transplantation centers to report additional cases. The potential clinical usefu[ ness of screening SOT candidates for MBL deficiency in relation to infectious complications after transplantation remains to be determined.

Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR.

Avidity of Ag recognition by tumor-specific T cells is one of the main parameters that determines the potency of a tumor rejection Ag. In this study we show that the relative efficiency of staining of tumor Ag-specific T lymphocytes with the corresponding fluorescent MHC class I/peptide multimeric complexes can considerably vary with staining conditions and does not necessarily correlate with avidity of Ag recognition. Instead, we found a clear correlation between avidity of Ag recognition and the stability of MHC class I/peptide multimeric complexes interaction with TCR as measured in dissociation kinetic experiments. These findings are relevant for both identification and isolation of tumor-reactive CTL.

HTPSELEX--a database of high-throughput SELEX libraries for transcription factor binding sites.

HTPSELEX is a public database providing access to primary and derived data from high-throughput SELEX experiments aimed at characterizing the binding specificity of transcription factors. The resource is primarily intended to serve computational biologists interested in building models of transcription factor binding sites from large sets of binding sequences. The guiding principle is to make available all information that is relevant for this purpose. For each experiment, we try to provide accurate information about the protein material used, details of the wet lab protocol, an archive of sequencing trace files, assembled clone sequences (concatemers) and complete sets of in vitro selected protein-binding tags. In addition, we offer in-house derived binding sites models. HTPSELEX also offers reasonably large SELEX libraries obtained with conventional low-throughput protocols. The FTP site contains the trace archives and database flatfiles. The web server offers user-friendly interfaces for viewing individual entries and quality-controlled download of SELEX sequence libraries according to a user-defined sequencing quality threshold. HTPSELEX is available from ftp://ftp.isrec.isb-sib.ch/pub/databases/htpselex/ and http://www.isrec.isb-sib.ch/htpselex.

Constant coefficient tests for random coefficient regression

Random coefficient regression models have been applied in differentfields and they constitute a unifying setup for many statisticalproblems. The nonparametric study of this model started with Beranand Hall (1992) and it has become a fruitful framework. In thispaper we propose and study statistics for testing a basic hypothesisconcerning this model: the constancy of coefficients. The asymptoticbehavior of the statistics is investigated and bootstrapapproximations are used in order to determine the critical values ofthe test statistics. A simulation study illustrates the performanceof the proposals.

A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis.

Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as ω-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell.

How binding are legal limits? Transitions from temporary to permanent work in Spain

This paper studies the duration pattern of xed-term contracts and the determinantsof their conversion into permanent ones in Spain, where the share of xed-termemployment is the highest in Europe. We estimate a duration model for temporaryemployment, with competing risks of terminating into permanent employment versusalternative states, and exible duration dependence. We nd that conversion rates aregenerally below 10%. Our estimated conversion rates roughly increase with tenure,with a pronounced spike at the legal limit, when there is no legal way to retain theworker on a temporary contract. We argue that estimated di¤erences in conversionrates across categories of workers can stem from di¤erences in worker outside optionsand thus the power to credibly threat to quit temporary jobs.

Patterns of calcium-binding proteins support parallel and hierarchical organization of human auditory areas.

The human primary auditory cortex (AI) is surrounded by several other auditory areas, which can be identified by cyto-, myelo- and chemoarchitectonic criteria. We report here on the pattern of calcium-binding protein immunoreactivity within these areas. The supratemporal regions of four normal human brains (eight hemispheres) were processed histologically, and serial sections were stained for parvalbumin, calretinin or calbindin. Each calcium-binding protein yielded a specific pattern of labelling, which differed between auditory areas. In AI, defined as area TC [see C. von Economo and L. Horn (1930) Z. Ges. Neurol. Psychiatr.,130, 678-757], parvalbumin labelling was dark in layer IV; several parvalbumin-positive multipolar neurons were distributed in layers III and IV. Calbindin yielded dark labelling in layers I-III and V; it revealed numerous multipolar and pyramidal neurons in layers II and III. Calretinin labelling was lighter than that of parvalbumin or calbindin in AI; calretinin-positive bipolar and bitufted neurons were present in supragranular layers. In non-primary auditory areas, the intensity of labelling tended to become progressively lighter while moving away from AI, with qualitative differences between the cytoarchitectonically defined areas. In analogy to non-human primates, our results suggest differences in intrinsic organization between auditory areas that are compatible with parallel and hierarchical processing of auditory information.

Constant interest rate projections without the curse of indeterminacy

Constant interest rate (CIR) projections are often criticized on the grounds that they are inconsistent with the existence of a unique equilibrium in a variety of forward-looking models. This note shows howto construct CIR projections that are not subject to that criticism, using a standard New Keynesian model as a reference framework.

Functional analysis of human POT1 and RPA : insights into the role of single stranded DNA binding proteins at telomeres

Abstract Telomeres, the natural ends of chromosomes, need to be protected from chromosome end fusions, aberrant homologous recombination and degradation. In humans, chromosome ends are specified through arrays of tandemly repeated 5'-TTAGGG-3' hexamers, ending in a 3' overhang. A complex formed by the six proteins TRF1, TRF2, hRap1, TIN2, TPP1 and POT1 specifically assocìates with and protects telomeres. Telomeres are maintained by semiconservative DNA replication and by a specialized reverse transcriptase, telomerase, that carries an RNA subunit which templates new telomeric repeat synthesis. The telomeric single stranded (ss) DNA binding protein POT1 protects the telomeric 3' overhang and modulates telomerase-mediated telomere elongation. It is possible that POT1 also influences DNA synthesis during semiconservative DNA replication, which is initiated by the DNA polymerase alpha-primase complex. The heterotrimeric ss DNA-binding protein RPA plays essential roles during DNA replication. RPA binds to ss DNA with high affinity in order to stabilize ss DNA and facilitate nascent strand synthesis at the replication fork. Here we investigate how the two proteins RPA and POT1 contribute to telomere maintenance by regulating semi-conservative DNA replication and telomerase. Using chromatin immunoprecipitation experiments, we show that RPA associates with telomeres during S-phase. Analysis of telomere structure in cells shRNA-depleted for RPA and POT1 reveals that loss of RPA and POT1 causes exposure of single-stranded DNA at telomeres, suggestive of incomplete DNA replication. Biochemical experiments using purified recombinant POT1 and RPA show that saturating telomeric oligonucleotides with POT1 or RPA reduces the primase activity of the DNA polymerase alpha-primase complex and the overall activity of telomerase. POT1 and RPA also increase the primer extension by DNA polymerase alpha-primase complex and the processivity of telomerase under certain conditions, although POT1 increases the activities to a greater extent than RPA. We propose that POT1 is required for proper replication of the lagging strand of telomeres and that some phenotypes observed in POT1-depleted cells may stern from incomplete DNA replication rather than de-protection of the single-stranded overhang. Résumé Les télomères, les extrémités normales des chromosomes linéaires, doivent être protégés des fusions chromosomiques, d'événements de recombinaison homologue aberrants et de phénomènes de dégradation. Chez l'Homme, les extrémités des chromosomes sont constitués d'ADN double brin répétitif de séquence 5'-TTAGGG-3', d'une extension simple brin 3' sortante et d'un complexe protéique formé des six facteurs TRF1, TRF2, hRap1, TIN2, TPP1 et POT1 qui, s'associant à cette séquence, protègent l'ADN télomèrique. Les télomères sont maintenus par la télomérase, une transcriptase inverse capable d'allonger l'extension 3' sortante télomérique. POT1 lie l'ADN simple brin télomérique et module l'élongation des télomères par la télomérase. POT1 pourrait en théorie également influencer la réplication semi-conservative de l'ADN. L'ADN-polymérase Pal alpha-primase amorce et initie la synthèse d'ADN. Pendant la réplication, l'ADN simple brin est stabilisé par RPA, un complexe hétérotrimèrique qui lie l'ADN simple brin. RPA facilite la synthèse du brin naissant à la fourche de réplication. Ici nous avons étudié comment ces deux protéines qui lient l'ADN simple brin, RPA et POT1, régulent la réplication des télomères par la télomérase et la machinerie classique de réplication de l'ADN. Par immunoprécipitation de chromatine (ChIP), nous montrons que RPA est localisé aux télomères lors de la phase S du cycle cellulaire. De plus, l'analyse de la structure des télomeres indique que !a perte de RPA ou de POT1 conduit à l'apparition d'ADN simple brin télomérique, suggérant une réplication incomplète de l'ADN télomérique in vivo. Par une approche complémentaire biochimique utilisant les protéines POT1 et RPA recombinantes purifiées, nous montrons également que la liaison de POT1 ou de RPA à des oligonucléotides télomériques bloque l'activité primase du complexe polymérase alpha/primase et réduit l'activité télomérase sur ces substrats. En revanche, leur liaison augmente l'activité ADN-polymérase du complexe polymérase alpha/primase, ainsi que fa processivité de la télomérase dans certaines conditions, POT1 étant le plus efficace des deux facteurs. Nous proposons que POT1 est nécessaire à la réplication du brin retardé au niveau des télomères, ce qui suggère que certains phénotypes des cellules déplétés en POT1 puissent résulter d'une réplication incomplète de l'ADN télémétrique plutôt que d'une déprotection de l'extrémité sortante des télomères.

Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin.

Cloning of a cDNA encoding a plasma membrane-associated, uronide binding phosphoprotein with physical properties similar to viral movement proteins.

Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication.