Neuropeptide Y: a missing link?

Yet another 'orphan' molecule that had to find its place in life after isolation and sequencing, neuropeptide Y appears to be an important cardiovascular neuroregulator and also links the sympathetic and renin-angiotensin systems. The peptide's physiologic and pathophysiologic roles, as well as its potential therapeutic value, are examined.

Caveolin-1 opens endothelial cell junctions by targeting catenins.

AIMS: A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown.¦METHODS AND RESULTS: Impedance spectroscopy, biochemistry, and morphology were used to investigate the role of caveolin-1 in the regulation of thrombin-induced opening of cell junctions in cultured human and mouse endothelial cells. Here, we demonstrate that the vascular endothelial (VE) cadherin/catenin complex targets caveolin-1 to endothelial cell junctions. Association of caveolin-1 with VE-cadherin/catenin complexes is essential for the barrier function decrease in response to the pro-inflammatory mediator thrombin, which causes a reorganization of the complex in a rope ladder-like pattern accompanied by a loss of junction-associated actin filaments. Mechanistically, we show that in response to thrombin stimulation the protease-activated receptor 1 (PAR-1) causes phosphorylation of caveolin-1, which increasingly associates with β- and γ-catenin. Consequently, the association of β- and γ-catenin with VE-cadherin is weakened, thus allowing junction reorganization and a decrease in barrier function. Thrombin-induced opening of cell junctions is lost in caveolin-1-knockout endothelial cells and after expression of a Y/F-caveolin-1 mutant but is completely reconstituted after expression of wild-type caveolin-1.¦CONCLUSION: Our results highlight the pivotal role of caveolin-1 in VE-cadherin-mediated cell adhesion via catenins and, in turn, in barrier function regulation.

Unexpected Role for Connexin37 in Circulating FVIII and Coagulation

The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. Cx37 is expressed in endothelial cells, platelets and megakaryocytes. We have recently shown that Cx37 limits thrombus propensity by permitting intercellular signaling between aggregating platelets. Here, we have performed high throughput phage display to identify potential binding partners for the regulatory intracellular C-terminus of Cx37 (Cx37CT). We retrieved 2 consensus binding motifs for Cx37CT: WHK...[K,R]XP... and FH-K...[K,R]XXP.... Sequence alignment against the NCBI protein database indicated 66% homology of one the selected peptides with FVIII B-domain. We performed cross-linking reactions using BS3 and confirmed that an 11-mer peptide of the FVIII B-domain sequence linked to recombinant Cx37CT. In vitro binding of this peptide to Cx37CT was also confirmed by surface plasmon resonance. The dissociation constant of FVIII B-domain peptides to Cx37CT was ~20 uM. Other peptide sequences, designed upstream or downstream of the FVIII B-domain sequence, showed very low or no affinity for Cx37CT. Finally, in vivo studies revealed that thrombin generation in platelet-poor plasma from Cx37-/- mice (endogenous thrombin potential: 634±11 nM min, mean±SEM) was increased compared to Cx37+/+ mice (427±12, P<0.001). Moreover, partial activated thromboplastin time (aPTT) was shorter in Cx37-/- (39.7±1.5 s) than in Cx37+/+ mice (45.9±1.8, P=0.03), whereas prothrombin time was comparable. The shorter aPTT in Cx37-/- mice correlated with higher circulating FVIII activity (46.0±0.7 vs. 53.5±2.7 s for Cx37+/+, P=0.03). Overall, our data show for the first time a functional interaction between FVIII and Cx37. This interaction may be relevant for the control of FVIII secretion and, thereby, in the regulation of levels of FVIII circulating in blood. In addition, these results may open new perspectives to improve the efficiency of recombinant FVIII manufacturing.

Citrobacter rodentium Requires Intestinal Neural Wiskott-Aldrich Syndrome Protein (N-WASp) for Actin Pedestal Formation, Colonization and Epithelial Barrier Disruption In Vivo

Background: Citrobacter rodentium is a natural mouse pathogen that is genetically closelyrelated to the human enteric pathogens enteropathogenic and enterohemorrhagic E. coli.Among the repertoire of conserved virulence factors that these pathogens deliver via typeIII secretion, Tir and EspF are responsible for the formation of characteristic actin-richpedestals and disruption of tight junction integrity, respectively. There is evidence In Vitrothese effectors accomplish this, at least in part, by subverting the normal host cellularfunctions of N-WASP, a critical regulator of branched chain actin assembly. Although NWASPhas been shown to be involved in pedestal formation In Vitro, the requirements ofN-WASP-mediated actin pedestals for intestinal colonization by attaching/effacing (A/E)pathogens In Vivo is not known. Furthermore, it is not known whether N-WASP is requiredfor EspF-mediated tight junction disruption. Methods: To investigate the role of N-WASPin the gut epithelium, we generated mice with intestine-specific deletion of N-WASP(iNWKO), by mating mice homozygous for a floxed N-WASP allele (N-WASPL2L/L2L) tomice expressing Cre recombinase under the villin promoter. Separately housed groups ofWT and iNWKO mice were inoculated with 5x108 GFP-expressing C. rodentium by intragastriclavage. Stool was collected 2, 4, 7, and 12 days after infection, and recoverablecolony forming units (CFUs) of C. rodentium were quantified by plating serial dilutions ofhomogenized stool on MacConkey's agar. GFP+ colonies were counted after 24 hoursincubation at 37°C. The presence of actin pedestals was investigated by electron microscopy(EM), and tight junction morphology was assessed by immunofluorescence staining ofoccludin, ZO-1 and claudin-2. Results: C. rodentium infection did not result in mortalityin WT or iNWKO mice. Compared to controls, iNWKO mice exhibited higher levels ofbacterial shedding during the first 4 days of infection (day 4 average: WT 5.2x104 CFU/gvs. iNWKO 4.7x105 CFU/g, p=0.08), followed by a more rapid clearance of C. rodentium, (day7-12 average: WT 2x106 CFU/g vs. iNWKO 2.7x105, p=0.01). EM and immunofluorescencerevealed the complete lack of actin pedestals in iNWKO mice and no mucosa-associatedGFP+ C. rodentium by day 7. WT controls exhibited tight junction disruption, reflected byaltered distribution of ZO-1, whereas iNWKO mice had no change in the pattern of ZO-1.Conclusion: Intestinal N-WASP is required for actin pedestal formation by C. rodentium InVivo, and ablation of N-WASP is associated with more rapid bacterial clearance and decreasedability of C. rodentium to disrupt intercellular junctions.

Fondaparinux for the treatment of superficial-vein thrombosis in the legs.

BACKGROUND: The efficacy and safety of anticoagulant treatment for patients with acute, symptomatic superficial-vein thrombosis in the legs, but without concomitant deep-vein thrombosis or symptomatic pulmonary embolism at presentation, have not been established. METHODS: In a randomized, double-blind trial, we assigned 3002 patients to receive either fondaparinux, administered subcutaneously at a dose of 2.5 mg once daily, or placebo for 45 days. The primary efficacy outcome was a composite of death from any cause or symptomatic pulmonary embolism, symptomatic deep-vein thrombosis, or symptomatic extension to the saphenofemoral junction or symptomatic recurrence of superficial-vein thrombosis at day 47. The main safety outcome was major bleeding. The patients were followed until day 77. RESULTS: The primary efficacy outcome occurred in 13 of 1502 patients (0.9%) in the fondaparinux group and 88 of 1500 patients (5.9%) in the placebo group (relative risk reduction with fondaparinux, 85%; 95% confidence interval [CI], 74 to 92; P<0.001). The incidence of each component of the primary efficacy outcome was significantly reduced in the fondaparinux group as compared with the placebo group, except for the outcome of death (0.1% in both groups). The rate of pulmonary embolism or deep-vein thrombosis was 85% lower in the fondaparinux group than in the placebo group (0.2% vs. 1.3%; 95% CI, 50 to 95; P<0.001). Similar risk reductions were observed at day 77. A total of 88 patients would need to be treated to prevent one instance of pulmonary embolism or deep-vein thrombosis. Major bleeding occurred in one patient in each group. The incidence of serious adverse events was 0.7% with fondaparinux and 1.1% with placebo. CONCLUSIONS: Fondaparinux at a dose of 2.5 mg once a day for 45 days was effective in the treatment of patients with acute, symptomatic superficial-vein thrombosis of the legs and did not have serious side effects. (Funded by GlaxoSmithKline; ClinicalTrials.gov number, NCT00443053.)

International Society of Urological Pathology (ISUP) Consensus Conference on Handling and Staging of Radical Prostatectomy Specimens. Working group 4: seminal vesicles and lymph nodes.

The 2009 International Society of Urological Pathology Consensus Conference in Boston made recommendations regarding the standardization of pathology reporting of radical prostatectomy specimens. Issues relating to the infiltration of tumor into the seminal vesicles and regional lymph nodes were coordinated by working group 4. There was a consensus that complete blocking of the seminal vesicles was not necessary, although sampling of the junction of the seminal vesicles and prostate was mandatory. There was consensus that sampling of the vas deferens margins was not obligatory. There was also consensus that muscular wall invasion of the extraprostatic seminal vesicle only should be regarded as seminal vesicle invasion. Categorization into types of seminal vesicle spread was agreed by consensus to be not necessary. For examination of lymph nodes, there was consensus that special techniques such as frozen sectioning were of use only in high-risk cases. There was no consensus on the optimal sampling method for pelvic lymph node dissection specimens, although there was consensus that all lymph nodes should be completely blocked as a minimum. There was also a consensus that a count of the number of lymph nodes harvested should be attempted. In view of recent evidence, there was consensus that the diameter of the largest lymph node metastasis should be measured. These consensus decisions will hopefully clarify the difficult areas of pathological assessment in radical prostatectomy evaluation and improve the concordance of research series to allow more accurate assessment of patient prognosis.

Effects of muscle fibre shortening on the characteristics of surface motor unit potentials.

Traditionally, studies dealing with muscle shortening have concentrated on assessing its impact on conduction velocity, and to this end, electrodes have been located between the end-plate and tendon regions. Possible morphologic changes in surface motor unit potentials (MUPs) as a result of muscle shortening have not, as yet, been evaluated or characterized. Using a convolutional MUP model, we investigated the effects of muscle shortening on the shape, amplitude, and duration characteristics of MUPs for different electrode positions relative to the fibre-tendon junction and for different depths of the MU in the muscle (MU-to-electrode distance). It was found that the effects of muscle shortening on MUP morphology depended not only on whether the electrodes were between the end-plate and the tendon junction or beyond the tendon junction, but also on the specific distance to this junction. When the electrodes lie between the end-plate and tendon junction, it was found that (1) the muscle shortening effect is not important for superficial MUs, (2) the sensitivity of MUP amplitude to muscle shortening increases with MU-to-electrode distance, and (3) the amplitude of the MUP negative phase is not affected by muscle shortening. This study provides a basis for the interpretation of the changes in MUP characteristics in experiments where both physiological and geometrical aspects of the muscle are varied.

A large bullet in the bladder.

All manner of foreign bodies have been extracted from the bladder. Introduction into the bladder may be through self-insertion, iatrogenic means or migration from adjacent organs. Extraction should be tailored according to the nature of the foreign body and should minimise bladder and urethral trauma. We report a case of a bullet injury to the bladder, which finally presented as a gross hematuria after remaining asymptomatic for four years. We present here an alternative to suprapubic cystostomy with a large bladder foreign body treated via a combined transurethral unroofing followed by removal using a grasper passed through a suprapubic laparoscopic port.

Schizotypal perceptual aberrations of time: correlation between score, behavior and brain activity.

A fundamental trait of the human self is its continuum experience of space and time. Perceptual aberrations of this spatial and temporal continuity is a major characteristic of schizophrenia spectrum disturbances--including schizophrenia, schizotypal personality disorder and schizotypy. We have previously found the classical Perceptual Aberration Scale (PAS) scores, related to body and space, to be positively correlated with both behavior and temporo-parietal activation in healthy participants performing a task involving self-projection in space. However, not much is known about the relationship between temporal perceptual aberration, behavior and brain activity. To this aim, we composed a temporal Perceptual Aberration Scale (tPAS) similar to the traditional PAS. Testing on 170 participants suggested similar performance for PAS and tPAS. We then correlated tPAS and PAS scores to participants' performance and neural activity in a task of self-projection in time. tPAS scores correlated positively with reaction times across task conditions, as did PAS scores. Evoked potential mapping and electrical neuroimaging showed self-projection in time to recruit a network of brain regions at the left anterior temporal cortex, right temporo-parietal junction, and occipito-temporal cortex, and duration of activation in this network positively correlated with tPAS and PAS scores. These data demonstrate that schizotypal perceptual aberrations of both time and space, as reflected by tPAS and PAS scores, are positively correlated with performance and brain activation during self-projection in time in healthy individuals along the schizophrenia spectrum.

A developmental framework for endodermal differentiation and polarity.

The endodermis is a root cell layer common to higher plants and of fundamental importance for root function and nutrient uptake. The endodermis separates outer (peripheral) from inner (central) cell layers by virtue of its Casparian strips, precisely aligned bands of specialized wall material. Here we reveal that the membrane at the Casparian strip is a diffusional barrier between the central and peripheral regions of the plasma membrane and that it mediates attachment to the extracellular matrix. This membrane region thus functions like a tight junction in animal epithelia, although plants lack the molecular modules that establish tight junction in animals. We have also identified a pair of influx and efflux transporters that mark both central and peripheral domains of the plasma membrane. These transporters show opposite polar distributions already in meristems, but their localization becomes refined and restricted upon differentiation. This "central-peripheral" polarity coexists with the apical-basal polarity defined by PIN proteins within the same cells, but utilizes different polarity determinants. Central-peripheral polarity can be already observed in early embryogenesis, where it reveals a cellular polarity within the quiescent center precursor cell. A strict diffusion block between polar domains is common in animals, but had never been described in plants. Yet, its relevance to endodermal function is evident, as central and peripheral membranes of the endodermis face fundamentally different root compartments. Further analysis of endodermal transporter polarity and manipulation of its barrier function will greatly promote our understanding of plant nutrition and stress tolerance in roots.

Fanconi anemia and genome stability : the role of FANCM

RÉSUMÉ: Le génome de toute cellule est susceptible d'être attaqué par des agents endogènes et exogènes. Afin de préserver l'intégrité génomique, les cellules ont développé des multitudes de mécanismes. La réplication de l'ADN, une étape importante durant le cycle cellulaire, constitue un stress et présente un danger important pour l'intégrité du génome. L'anémie de Fanconi est une maladie héréditaire rare dont les protéines impliquées semblent jouer un rôle crucial dans la réponse au stress réplicatif. La maladie est associée à une instabilité chromosomique ainsi qu'à une forte probabilité de développer des cancers. Les cellules des patients souffrant de l'anémie de Fanconi sont sensibles à des agents interférant avec la réplication de l'ADN, et plus particulièrement àdes agents qui fient les deux brins d'ADN d'une manière covalente. L'anémie de Fanconi est une maladie génétiquement hétérogène. Treize protéines ont pu être identifiées. Elles semblent figurer dans une même voie de signalisation qui est aussi connue sous le nom de « FA/BRCA pathway », car un des gènes est identique au gène BRCA2 (breast cancer susceptibility gene 2). Huit protéines forment un complexe nucléaire dont l'intégrité est nécessaire à la monoubiquitination de deux autres protéines, FANCD2 et FANCI, en réponse à un stress réplicatif. A ce jour, la fonction moléculaire des protéines du « FA/BRCA pathway »reste encore mal décrite. Au début de mon travail de thèse, nous avons donc décidé de purifier les protéines du complexe nucléaire et d'étudier leurs propriétés biochimiques. Nous avons tout d'abord étudié les cinq protéines connues à l'époque qui sont FANCA, FANCC, FANCE, FANCF et FANCG. Par la suite, nous avons étendu notre étude à des protéines découvertes plus récemment, FANCL, FANCM et FAAP24, en concentrant finalement notre travail sur la caractérisation de FANCM. FANCM, contrairement aux autres protéines du complexe, est constituée de deux domaines conservés suggérant un rôle important dans le métabolisme de l'ADN. Il s'agit d'un domaine « DEAH box hélicase »situé dans la partie N-terminale et d'un domaine « ERCC4 nuclease »situé dans la partie C-terminale de la protéine. Dans cette étude, nous avons purifié avec succès la protéine FANCM entière à partir d'un système hétérologue. Nous montrons que FANCM s'attache de manière spécifique à des jonctions de Holliday et des fourches de réplication. De plus, nous démontrons que FANCM peut déplacer le point de jonction de ces structures via son domaine hélicase de manière dépendante de l'ATP. FANCM est aussi capable de dissocier de grands intermédiaires de la recombinaison, via la migration de jonctions de Holliday à travers une région d'homologie de 2.6 kb. Tous ces résultats suggèrent que FANCM peut s'attacher spécifiquement à des fourches de réplication et à des jonctions de Holliday in vitro et que son domaine hélicase est associé à une activité migratoire efficace. Nous pensons que FANCM peut avoir un rôle direct sur les intermédiaires de réplication. Ceci est en accord avec l'idée que les protéines de l'anémie de Fanconi coordonnent la réparation de l'ADN au niveau des fourches de réplication arrêtées. Nos résultats donnent une première indication quant au rôle de FANCM dans la cellule et peuvent contribuer à élucider la fonction de cette voie de signalisation peu comprise jusqu'à présent. SUMMARY: The genome of every cell is subject to a constant offence by endogenous and exogenous agents. Not surprisingly; cells have evolved a multitude of mechanisms which aim at preserving genomic integrity. A key step during the life cycle of a cell, DNA replication itself, constitutes a special danger to the integrity of the genome. The proteins defective in the rare hereditary disease Fanconi anemia (FA) are suspected to play a crucial role in the cellular response to DNA replication stress. The disease is associated with chromosomal instability and pronounced cancer susceptibility. Cells from Fanconi anemia patients are sensitive to a variety of agents which interfere with DNA replication, DNA interstrand cross-linking agents being particularly threatening to their survival. Fanconi anemia is a genetically heterogeneous disease with 13 different proteins identified, which seem to work together in a common pathway. Since one of the FA genes is identical to the breast cancer susceptibility gene BRCA2, it is also referred to as the FA/BRCA pathway. Eight proteins form a nuclear complex, whose integriry is required for the monoubiquitination of two other FA proteins, FANCD2 and FANCI, in response to DNA replication stress. Despite intensive research, the function of the FA/BRCA pathway at a molecular level has remained largely elusive so far. At the beginning of my thesis, we therefore decided to purify the proteins of the FA core complex and to investigate their biochemical properties. We started with the five proteins which were known at that time, FANCA, FANCC, FANCE, FANCF, and FACG. Later on, we extended our studies to the newly discovered proteins FANCL, FANCM, and FAAP24, and eventually focused our work on the characterisation of FANCM. In contrast to the other core complex proteins, FANCM contains two conserved domains, which point to a role in DNA metabolism: an N-terminal DEAH box helicase domain and a C-terminal ERCC4 nuclease domain. In this study, we have successfully purified full-length FANCM from a recombinant source. We show that purified FANCM binds to branched DNA molecules, such as Holliday junctions and replication forks, with high specificity and affinity. In addition, we demonstrate that FANCM can translocate the junction point of branched DNA molecules due to its helicase domain in an ATPase-dependent manner. FANCM can even dissociate large recombination intermediates, via branch migration of Holliday junctions through a 2.6 kb region of homology. Taken together, our data suggest that FANCM can specifically bind to replication forks and Holliday junctions in vitro, and that its DEAH box helicase domain is associated with a potent branch migration activity. We propose that FANCM might have a direct role in the processing of DNA replication intermediates. This is consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks. Our findings provide a first hint as to the context in which FANCM might play a role in the cell. We are optimistic that they might be key to further elucidate the function of a pathway which is far from being understood.

Microbial communities in a porphyry copper tailings impoundment and their impact on the geochemical dynamics of the mine waste

The distribution and diversity of acidophilic bacteria of a tailings impoundment at the La Andina copper mine, Chile, was examined. The tailings have low sulfide (1.7% pyrite equivalent) and carbonate (1.4% calcite equivalent) contents and are stratified into three distinct zones: a surface (0-70-80 cm) `oxidation zone' characterized by low-pH (2.5-4), a `neutralization zone' (70-80 to 300-400 cm) and an unaltered `primary zone' below 400 cm. A combined cultivation-dependent and biomolecular approach (terminal restriction enzyme fragment length polymorphism and 16S rRNA clone library analysis) was used to characterize the indigenous prokaryotic communities in the mine tailings. Total cell counts showed that the microbial biomass was greatest in the top 125 cm of the tailings. The largest numbers of bacteria (10(9) g(-1) dry weight of tailings) were found at the oxidation front (the junction between the oxidation and neutralization zones), where sulfide minerals and oxygen were both present. The dominant iron-/sulfur-oxidizing bacteria identified at the oxidation front included bacteria of the genus Leptospirillum (detected by molecular methods), and Gram-positive iron-oxidizing acidophiles related to Sulfobacillus (identified both by molecular and cultivation methods). Acidithiobacillus ferrooxidans was also detected, albeit in relatively small numbers. Heterotrophic acidophiles related to Acidobacterium capsulatum were found by molecular methods, while another Acidobacterium-like bacterium and an Acidiphilium sp. were isolated from oxidation zone samples. A conceptual model was developed, based on microbiological and geochemical data derived from the tailings, to account for the biogeochemical evolution of the Piuquenes tailings impoundment.

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis.

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.

Subjective mental time: the functional architecture of projecting the self to past and future.

Abstract Human experience takes place in the line of mental time (MT) created through 'self-projection' of oneself to different time-points in the past or future. Here we manipulated self-projection in MT not only with respect to one's life events but also with respect to one's faces from different past and future time-points. Behavioural and event-related functional magnetic resonance imaging activity showed three independent effects characterized by (i) similarity between past recollection and future imagination, (ii) facilitation of judgements related to the future as compared with the past, and (iii) facilitation of judgements related to time-points distant from the present. These effects were found with respect to faces and events, and also suggest that brain mechanisms of MT are independent of whether actual life episodes have to be re-experienced or pre-experienced, recruiting a common cerebral network including the anteromedial temporal, posterior parietal, inferior frontal, temporo-parietal and insular cortices. These behavioural and neural data suggest that self-projection in time is a fundamental aspect of MT, relying on neural structures encoding memory, mental imagery and self.

Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus.

We have recently reported that Notch 1, a member of the Notch multigene family, is essential for the development of murine T cells. Using a mouse model in which Notch 1 is inactivated in bone marrow (BM) precursors we have shown that B cells instead of T cells are found in the thymus of BM chimeras. However, it is not clear whether these B cells develop by default from a common lymphoid precursor due to the absence of Notch 1 signaling, or whether they arise as a result of perturbed migration of BM-derived B cells and/or altered homeostasis of normal resident thymic B cells. In this report we show that Notch 1-deficient thymic B cells resemble BM B cells in phenotype and turnover kinetics and are located predominantly in the medulla and corticomedullary junction. Peripheral blood lymphocyte analysis shows no evidence of recirculating Notch1(-/)- BM B cells. Furthermore, lack of T cell development is not due to a failure of Notch1(-/)- precursors to home to the thymus, as even after intrathymic reconstitution with BM cells, B cells instead of T cells develop from Notch 1-deficient precursors. Taken together, these results provide evidence for de novo ectopic B cell development in the thymus, and support the hypothesis that in the absence of Notch 1 common lymphoid precursors adopt the default cell fate and develop into B cells instead.