The nuclear matrix: a critical appraisal.

It is becoming increasingly clear that the cell nucleus is a highly structurized organelle. Because of its tight compartmentalization, it is generally believed that a framework must exist, responsible for maintaining such a spatial organization. Over the last twenty years many investigations have been devoted to identifying the nuclear framework. Structures isolated by different techniques have been obtained in vitro and are variously referred to as nuclear matrix, nucleoskeleton or nuclear scaffold. Many different functions, such as DNA replication and repair, mRNA transcription, processing and transport have been described to occur in close association with these structures. However, there is still much debate as to whether or not any of these preparations corresponds to a nuclear framework that exists in vivo. In this article we summarize the most commonly-used methods for obtaining preparations of nuclear frameworks and we also stress the possible artifacts that can be created in vitro during the isolation procedures. Emphasis is placed also on the protein composition of the frameworks as well as on some possible signalling functions that have been recently described to occur in tight association with the nuclear matrix.

T cell receptor specificity is critical for the development of epidermal gammadelta T cells.

A particular feature of gammadelta T cell biology is that cells expressing T cell receptor (TCR) using specific Vgamma/Vdelta segments are localized in distinct epithelial sites, e.g., in mouse epidermis nearly all gammadelta T cells express Vgamma3/Vdelta1. These cells, referred to as dendritic epidermal T cells (DETC) originate from fetal Vgamma3+ thymocytes. The role of gammadelta TCR specificity in DETC's migration/localization to the skin has remained controversial. To address this issue we have generated transgenic (Tg) mice expressing a TCR delta chain (Vdelta6.3-Ddelta1-Ddelta2-Jdelta1-Cdelta), which can pair with Vgamma3 in fetal thymocytes but is not normally expressed by DETC. In wild-type (wt) Vdelta6.3Tg mice DETC were present and virtually all of them express Vdelta6.3. However, DETC were absent in TCR-delta(-/-) Vdelta6.3Tg mice, despite the fact that Vdelta6.3Tg gammadelta T cells were present in normal numbers in other lymphoid and nonlymphoid tissues. In wt Vdelta6.3Tg mice, a high proportion of in-frame Vdelta1 transcripts were found in DETC, suggesting that the expression of an endogenous TCR-delta (most probably Vdelta1) was required for the development of Vdelta6.3+ epidermal gammadelta T cells. Collectively our data demonstrate that TCR specificity is essential for the development of gammadelta T cells in the epidermis. Moreover, they show that the TCR-delta locus is not allelically excluded.

The tumour suppressor LATS2 interacts with both Signalosome and E3 ubiquitin ligases : implications in control of cell cycle progression

SUMMARY LATS2 is a member of the Lats tumour suppressor gene family. The human LATS2 gene is located at chromosome 13q11-12, which has been shown to be a hot spot (67%) for LOH in nonsmall cell lung cancer. Both lats mosaic flies and LATS1 deficient mice spontaneously develop tumours, an observation that is explained by the function of LATS1 in suppressing tumourigenesis by negatively regulating cell proliferation by modulating Cdc2/Cyclin A activity. LATS1 also plays a critical role in maintenance of ploidy through its action on the spindle assembly checkpoint. Initial insights into the function of LATS2 reveals that the protein is involved in the G2/M transition of the cell cycle, whereby it controls the phosphorylation status of Cdc25C. The aim of the present study was to identify LATS2 interacting partners that would provide a more thorough understanding of the molecular pathways in which the protein is involved. The yeast two-hybrid system identified a number of candidate genes that interact with LATS2. Most of the interactions were confirmed biochemically by GST-pull down assays that enabled us to demonstrate that LATS2 is an integral component of the Signalosome complex. The Signalosome is thought to be required for the establishment of functional Cullin-based E3 ubiquitin ligases, the substrate-recognition elements of the ubiquitin-mediated protein proteolytic pathway. The findings that LATS2 also interacts with all of the components of the E3 enzymes allows us to postulate that LATS2 is probably involved in the regulation of this Signalosome-E3 super-complex. In addition, the discovery that LATS2 associates with multiple protein kinases localised at the cellular membrane and in various signalling cascades supports the idea that LATS2 functions as an integrator of signals which allows it to monitor the activity of these pathways and translate these signals through its action on the Signalosome. Furthermore, the observation that a kinase-dead LATS2 mutant arrests at the G2/M phase of the cell cycle, demonstrates that the protein, through the action of its kinase domain, is crucial for progression through the cell cycle, an action in accordance to its proposed role as a regulator of E3 ubiquitin ligases. The findings presented herein provide evidence that LATS2 associates with the Signalosome-E3 ubiquitin ligases super-complex which governs protein stability. Any alteration of the protein would have a strong impact on pathways that modulate cell proliferation, as shown by its implication in tumourigenesis. RESUME LATS2 est un membre de la famille de gènes suppresseurs de tumeurs LATS. Le gène humain LATS2 est situé sur le chromosome 13q11-12, une région qui s'est avérée être un point sensible (67%) dans la perte d'hétérozigosité (LOH) notamment pour le cancer du poumon. Le fait que des tumeurs se développent spontanément chez les souris qui sont déficientes pour le gène LATS1 ainsi que dans des cellules mutantes pour LATS chez la Drosophile, est expliqué Par la fonction de LATS1, qui est de supprimer l'apparition de tumeurs en réprimant la prolifération cellulaire à travers sa capacité à réguler l'activité de Cdc2/Cyciine A. LATS1 joue également un rôle important au niveau du maintient de la ploïdie de la cellule, au travers de son action sur les points de contrôle de l'assemblage du fuseau mitotique. Les premières études du gène LATS2 indiquent que la protéine est, par son contrôle des réactions de phosphorylation de la Cdc25C, impliquée dans la transition 021M. Le but de cette étude était d'identifier les protéines qui interagissent avec LATS2, en vue d'obtenir une compréhension plus approfondie des mécanismes moléculaires dans lesquels LATS2 se trouve engagée. Le système de double-hybride chez la levure a permis l'identification d'un grand nombre de gènes qui interagissent avec LATS2. La plupart des interactions ont été confirmées par GST «pull clown», une technique in vitro qui a permis de démontrer que LATS2 est un composant intégral du Signalosome. Ce complexe est supposé réguler l'activité des E3 ubiquitine-rigases, les éléments responsables du recrutement des substrats qui doivent être recyclés par la voie de dégradation ubiquitine-dépendante. Les résultats obtenus indiquent également que LATS2 interagit avec tous les composants des enzymes E3, ce qui nous permet de soumettre l'idée selon laquelle la protéine LATS2 est en fait impliquée dans la régulation du complexe Signalosorne-E3. De plus, la découverte que LATS2 se trouve associée à plusieurs protéines kinases localisées au niveau de la membrane cellulaire, ainsi que dans diverses voies de transduction, confirment l'idée que LATS2 fonctionne en tant que molécule qui intègre les signaux en provenance de ces différentes voies cellulaires. De ce fait, il lui serait possible de coordonner la destruction des protéines au moyen du complexe Signalosome, permettant ainsi de réprimer l'activité des voies de signalisation. En outre, l'introduction d'une mutation dans le domaine kinase de LATS2 résulte en l'arrêt du cycle cellulaire en G2/M, ce qui montre que la protéine, au travers de son domaine kinase, est cruciale pour le bon fonctionnement du cycle cellulaire, ceci en accord avec son rôle proposé comme régulateur des E3 ubiquitine-ligases. Les résultats présentés dans ce manuscrit démontrent que la protéine LATS2 se trouve associée au complexe Signalosome-E3 qui régule la dégradation des protéines. La moindre modification de la protéine engendrerait des répercussions importantes au niveau des voies de transduction qui contrôlent fa prolifération ceilulaire, ce qui atteste du rôle déterminant que joue LAT32 dans la tumorigénèse.

Dynamic visual information plays a critical role for spatial navigation in water but not on solid ground.

In the Morris water maze (MWM) task, proprioceptive information is likely to have a poor accuracy due to movement inertia. Hence, in this condition, dynamic visual information providing information on linear and angular acceleration would play a critical role in spatial navigation. To investigate this assumption we compared rat's spatial performance in the MWM and in the homing hole board (HB) tasks using a 1.5 Hz stroboscopic illumination. In the MWM, rats trained in the stroboscopic condition needed more time than those trained in a continuous light condition to reach the hidden platform. They expressed also little accuracy during the probe trial. In the HB task, in contrast, place learning remained unaffected by the stroboscopic light condition. The deficit in the MWM was thus complete, affecting both escape latency and discrimination of the reinforced area, and was thus task specific. This dissociation confirms that dynamic visual information is crucial to spatial navigation in the MWM whereas spatial navigation on solid ground is mediated by a multisensory integration, and thus less dependent on visual information.

Treatment of large bone defects with the Ilizarov technique.

Between 1985 and 1990 we treated 11 large segmental bone defects (average 6.7 cm) in ten patients with the Ilizarov technique. Open fractures, type III according to Gustilo, represented the largest group (8 of 11 cases). The average delay before the Ilizarov technique was initiated was 8.9 months. The external fixator was usually maintained for 1 year. Bone regeneration was obtained in every case. Consolidation was not fulfilled with this technique in three cases. The complications observed were one refracture, four leg-length discrepancies (average 1.5 cm), and five axial deformities exceeding 5 degrees. No pin-track infection was observed. In our limited series of four type IIIC open fractures treated by the Ilizarov technique, no patients required amputation. The Ilizarov technique is particularly useful in the treatment of large bone defects, without major complications, especially if there is an adequate initial debridement.

Bcl9/Bcl9l are critical for Wnt-mediated regulation of stem cell traits in colon epithelium and adenocarcinomas.

Canonical Wnt signaling plays a critical role in stem cell maintenance in epithelial homeostasis and carcinogenesis. Here, we show that in the mouse this role is critically mediated by Bcl9/Bcl9l, the mammalian homologues of Legless, which in Drosophila is required for Armadillo/beta-catenin signaling. Conditional ablation of Bcl9/Bcl9l in the intestinal epithelium, where the essential role of Wnt signaling in epithelial homeostasis and stem cell maintenance is well documented, resulted in decreased expression of intestinal stem cell markers and impaired regeneration of ulcerated colon epithelium. Adenocarcinomas with aberrant Wnt signaling arose with similar incidence in wild-type and mutant mice. However, transcriptional profiles were vastly different: Whereas wild-type tumors displayed characteristics of epithelial-mesenchymal transition (EMT) and stem cell-like properties, these properties were largely abrogated in mutant tumors. These findings reveal an essential role for Bcl9/Bcl9l in regulating a subset of Wnt target genes involved in controlling EMT and stem cell-related features and suggest that targeting the Bcl9/Bcl9l arm of Wnt signaling in Wnt-activated cancers might attenuate these traits, which are associated with tumor invasion, metastasis, and resistance to therapy.

Macrophage migration inhibitory factor: a counter-regulator of glucocorticoid action and critical mediator of septic shock.

Recent studies have led to the discovery of a mediator that acts as an endogenous counter-regulator of glucocorticoid action within the immune system. Isolated as a product of anterior pituitary cells, this protein was found to have the sequence of macrophage migration inhibitory factor (MIF), one of the first cytokine activities to be described. Macrophages and T cells release MIF in response both to various inflammatory stimuli and upon incubation with low concentrations of glucocorticoids. The glucocorticoid-induced secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once secreted, MIF "overrides" the anti-inflammatory and immunosuppressive effects of steroids on macrophage and T-cell cytokine production. The physiological role of MIF thus appears to be to counter-balance steroid inhibition of the inflammatory response. Anti-MIF antibodies fully protect animals from experimentally induced gram-negative or gram-positive septic shock, an effect that may be the result of the increased anti-inflammatory effects of glucocorticoids after neutralization of endogenous MIF. Anti-MIF therapeutic strategies are presently under development and may prove to be a means to modulate cytokine production in septic shock as well as in other inflammatory disease states.

Effect of epinephrine on oxygen consumption and delivery during progressive hemorrhage.

OBJECTIVE: To determine whether, during hemorrhagic shock, the effect of epinephrine on energy metabolism could be deleterious, by enhancing the oxygen requirement at a given level of oxygen delivery (DO2). DESIGN: Prospective, randomized, control trial. SETTING: Experimental laboratory. SUBJECTS: Two groups of seven mongrel dogs were studied. The epinephrine group received a continuous infusion of epinephrine (1 microgram/min/kg) while the control group received saline. INTERVENTION: Dogs were anesthetized with pentobarbital, and shock was produced by stepwise hemorrhage. MEASUREMENTS AND MAIN RESULTS: Oxygen consumption (VO2) was continuously measured by the gas exchange technique, while DO2 was independently calculated from cardiac output (measured by thermodilution) and blood oxygen content. A dual-lines regression fit was applied to the DO2 vs. VO2 plot. The intersection of the two regression lines defined the critical value of DO2. Values above critical DO2 belonged to phase 1, while phase 2 occurred below critical DO2. In the control group, VO2 was independent of DO2 during phase 1; VO2 was dependent on DO2 during phase 2. In the epinephrine group, the expected increase in VO2 (+19%) and DO2 (+50%) occurred under normovolemic conditions. During hemorrhage, VO2 immediately decreased, and the slope of phase 1 was significantly (p < .01) different from zero, and was significantly (p < .05) steeper than in the control group (0.025 +/- 0.005 vs. 0.005 +/- 0.010). However, the critical DO2 (8.7 +/- 1.7 vs. 9.7 +/- 2.4 mL/min/kg), the critical VO2 (5.6 +/- 0.5 vs. 5.5 +/- 0.9 mL/min/kg), and the slope of phase 2 (0.487 +/- 0.080 vs. 0.441 +/- 0.130) were not different from control values. CONCLUSIONS: The administration of pharmacologic doses of epinephrine significantly increased VO2 under normovolemic conditions due to the epinephrine-induced thermogenic effect. This effect progressively decreased during hemorrhage. The critical DO2 and the relationship between DO2 and VO2 in the supply-dependent phase of shock were unaffected by epinephrine infusion. These results suggest that during hemorrhagic shock, epinephrine administration did not exert a detrimental effect on the relationship between DO2 and VO2.

CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation.

CARMA1 is a lymphocyte-specific member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which coordinate signaling pathways emanating from the plasma membrane. CARMA1 interacts with Bcl10 via its caspase-recruitment domain (CARD). Here we investigated the role of CARMA1 in T cell activation and found that T cell receptor (TCR) stimulation induced a physical association of CARMA1 with the TCR and Bcl10. We found that CARMA1 was constitutively associated with lipid rafts, whereas cytoplasmic Bcl10 translocated into lipid rafts upon TCR engagement. A CARMA1 mutant, defective for Bcl10 binding, had a dominant-negative (DN) effect on TCR-induced NF-kappa B activation and IL-2 production and on the c-Jun NH(2)-terminal kinase (Jnk) pathway when the TCR was coengaged with CD28. Together, our data show that CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation and CD28 costimulation-dependent Jnk activation.

Ablation of caterpillar labial salivary glands: technique for determining the role of saliva in insect-plant interactions.

There has been an ardent interest in herbivore saliva due to its roles in inducing plant defenses and its impact on herbivore fitness. Two techniques are described that inhibit the secretion of labial saliva from the caterpillar, Helicoverpa zea, during feeding. The methods rely on cauterizing the caterpillar's spinneret, the principal secretory structure of the labial glands, or surgically removing the labial salivary gland. Both methods successfully inhibit secretion of saliva and the principal salivary enzyme glucose oxidase. Caterpillars with inhibited saliva production feed at similar rates as the untreated caterpillars, pupate, and emerge as adults. Glucose oxidase has been suggested to increase the caterpillar's survival through the suppression of inducible anti-herbivore defenses in plants. Tobacco (Nicotiana tabacum) leaves fed on by caterpillars with ablated salivary glands had significantly higher levels of nicotine, an inducible anti-herbivore defense compound of tobacco, than leaves fed upon by caterpillars with intact labial salivary glands. Tomato (Lycopersicon esculentum) leaves fed upon by caterpillars with suppressed salivary secretions showed greatly reduced evidence of hydrogen peroxide formation compared to leaves fed upon by intact caterpillars. These two methods are useful techniques for determining the role that saliva plays in manipulating plant anti-herbivore defenses.

Identification of an expanded population of activated CD4(+) CD25(+) T cells expressing CD45RO and IL-7R in kidney transplant recipients

Recent studies have shown that CD4+ CD25+ T cells belong to two functionally different T lymphocytes, i.e. regulatory T cells (Treg) or activated T cells (Tact), which can be distinguished based on the expression of CD45RO and IL-7R: Treg (FoxP3+) are CD45RO+ IL-7R- , whereas Tact (FoxP3- ) are CD45RO+ IL- 7R+. In order to determine if a CD4+ CD25+ CD45RO+ IL-7R+ activated T cell population might be identified in kidney transplant recipients, we studied 27 healthy subjects (HS) and 23 kidney recipients, of whom 17 had stable graft function under standard immunosuppression (IS), 5 had biopsy-proven chronic humoral rejection (CHR), and one was a stable "tolerant" patient who had discontinued IS for more than 2 years. Phenotypical analysis by flow cytometry and functional assays by MLR were performed. Overall, the Tact population was found to be significantly increased in 87% of the transplant recipients (mean: 18.8±10.1% of CD4+ CD25+ T cells) compared to HS (mean: 4.5±2.0%; P<0.0001). In the 5 patients with CHR, this Tact population was highly expanded (31.3±9.3%; P<0.0001), whereas it was comparable to HS in the "tolerant" recipient (4.7%). Intermediate levels (16.0±6.9%; P<0.0001) were found in the 17 stable recipients. In CHR, the proliferative capacity of the Tact population was found to be 5-fold higher when stimulated by irradiated donor PBMC as compared to a stimulation by irradiated 3rd party PBMC. After kidney transplantation, an expanded circulating CD4+ CD25+ T cell population characterized by the expression of CD45RO and IL-7R was found in most recipients, particularly in those with CHR. In a patient with long-term operational tolerance, this Tact population was similar to HS. Measuring circulating Tact may become a useful monitoring tool after transplantation.

Revue critique d'instruments pour evaluer la douleur chez les personnes cérébrolésées non communicantes aux soins intensifs [Critical review of instruments to assess pain in the non communicative brain injured persons in intensive care].

The purpose of this review is to critically appraise the pain assessment tools for non communicative persons in intensive care available in the literature and to determine their relevance for those with brain injury. Nursing and medical electronic databases were searched to identify pain tools, with a description of psychometric proprieties, in English and French. Seven of the ten tools were considered relevant and systematically evaluated according to the criteria and the indicators in the following five areas: conceptualisation, target population, feasibility and clinical utility, reliability and validity. Results indicate a number of well designed pain tools, but additional work is necessary to establish their accuracy and adequacy for the brain injured non communicative person in intensive care. Recommendations are made to choose the best tool for clinical practice and for research.

Solid phase microextraction as a short-term sampling technique for BTEX occupational exposure

Solid phase microextraction (SPME) has been widely used for many years in various applications, such as environmental and water samples, food and fragrance analysis, or biological fluids. The aim of this study was to suggest the SPME method as an alternative to conventional techniques used in the evaluation of worker exposure to benzene, toluene, ethylbenzene, and xylene (BTEX). Polymethylsiloxane-carboxen (PDMS/CAR) showed as the most effective stationary phase material for sorbing BTEX among other materials (polyacrylate, PDMS, PDMS/divinylbenzene, Carbowax/divinylbenzene). Various experimental conditions were studied to apply SPME to BTEX quantitation in field situations. The uptake rate of the selected fiber (75 microm PDMS/CAR) was determined for each analyte at various concentrations, relative humidities, and airflow velocities from static (calm air) to dynamic (> 200 cm/s) conditions. The SPME method also was compared with the National Institute of Occupational Safety and Health method 1501. Unlike the latter, the SPME approach fulfills the new requirement for the threshold limit value-short term exposure limit (TLV-STEL) of 2.5 ppm for benzene (8 mg/m(3))