Cell viability and noninvasive in vivo MRI tracking of 3D cell encapsulating self-assembled microcontainers.

Several molecular therapies require the implantation of cells that secrete biotherapeutic molecules and imaging the location and microenvironment of the cellular implant to ascertain its function. We demonstrate noninvasive in vivo magnetic resonance imaging (MRI) of self-assembled microcontainers that are capable of cell encapsulation. Negative contrast was obtained to discern the microcontainer with MRI; positive contrast was obtained in the complete absence of background signal. MRI on a clinical scanner highlights the translational nature of this research. The microcontainers were loaded with cells that were dispersed in an extracellular matrix, and implanted both subcutaneously and in human tumor xenografts in SCID mice. MRI was performed on the implants, and microcontainers retrieved postimplantation showed cell viability both within and proximal to the implant. The microcontainers are characterized by their small size, three dimensionality, controlled porosity, ease of parallel fabrication, chemical and mechanical stability, and noninvasive traceability in vivo.

Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription.

The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAP(c), a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.

Full activation of the T cell receptor requires both clustering and conformational changes at CD3.

T cell receptor (TCR-CD3) triggering involves both receptor clustering and conformational changes at the cytoplasmic tails of the CD3 subunits. The mechanism by which TCRalphabeta ligand binding confers conformational changes to CD3 is unknown. By using well-defined ligands, we showed that induction of the conformational change requires both multivalent engagement and the mobility restriction of the TCR-CD3 imposed by the plasma membrane. The conformational change is elicited by cooperative rearrangements of two TCR-CD3 complexes and does not require accompanying changes in the structure of the TCRalphabeta ectodomains. This conformational change at CD3 reverts upon ligand dissociation and is required for T cell activation. Thus, our permissive geometry model provides a molecular mechanism that rationalizes how the information of ligand binding to TCRalphabeta is transmitted to the CD3 subunits and to the intracellular signaling machinery.

T cell receptor V beta repertoire in mice lacking endogenous mouse mammary tumor provirus.

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination.

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

MCL1 is deregulated in subgroups of diffuse large B-cell lymphoma.

Myeloid cell leukemia-1 (MCL1) is an anti-apoptotic member of the BCL2 family that is deregulated in various solid and hematological malignancies. However, its role in the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL) is unclear. We analyzed gene expression profiling data from 350 DLBCL patient samples and detected that activated B-cell-like (ABC) DLBCLs express MCL1 at significantly higher levels compared with germinal center B-cell-like DLBCL patient samples (P=2.7 × 10(-10)). Immunohistochemistry confirmed high MCL1 protein expression predominantly in ABC DLBCL in an independent patient cohort (n=249; P=0.001). To elucidate molecular mechanisms leading to aberrant MCL1 expression, we analyzed array comparative genomic hybridization data of 203 DLBCL samples and identified recurrent chromosomal gains/amplifications of the MCL1 locus that occurred in 26% of ABC DLBCLs. In addition, aberrant STAT3 signaling contributed to high MCL1 expression in this subtype. Knockdown of MCL1 as well as treatment with the BH3-mimetic obatoclax induced apoptotic cell death in MCL1-positive DLBCL cell lines. In summary, MCL1 is deregulated in a significant fraction of ABC DLBCLs and contributes to therapy resistance. These data suggest that specific inhibition of MCL1 might be utilized therapeutically in a subset of DLBCLs.

MOLECULAR MECHANISMS OF A ROOT SYSTEM TO SOIL ADAPTATION

Contrairement aux animaux, les plantes sont des organismes sessiles qui ne possèdent pas de mécanismes de fuite quand les conditions environnementales ne sont plus optimales. Les plantes sont physiquement ancrées à l'endroit où elles ont germées et aux conditions environnementales qui parfois peuvent être extrêmes. Les possibilités d'acclimatation de différentes espèces, parfois même de groupes de plantes au sein d'une même espèce, peuvent varier mais repose sur une adaptation génétique de la plante. L'adaptation est un long processus qui repose sur l'apparition spontanée de mutations génétiques, leur mise à l'épreuve face aux conditions environnementales, et dans le cas où la mutation a un impact positif sur la survie dans cet habitat particulier, elle sera maintenue dans une population donnée de plantes. De telles populations, appelées écotypes, sont le matériel de départ pour la découverte de gènes qui induisent un bénéfice pour la plante dans un environnement donné. La plante la plus étudiée en biologie moléculaire est Arabidopsis thaliana, l'arabette des prés. Dans une étude précédente, les racines d'écotypes naturels d'Arabidopsis ont été comparées et un écotype, Uk-1, avait le système racinaire le plus particulier. Cet écotype possède des racines beaucoup plus courtes et plus ramifiées que tous les autres écotypes. Des analyses plus poussées ont montré qu'une seule mutation dans un gène était la cause de ce phénotype, le gène BREVIS RADIX (BRX), mot latin signifiant 'racine courte'. Bien que l'on connaisse le gène BRX, on connaît finalement peu de choses sur son importance adaptative. Dans cette étude, nous avons montré que la mutation dans le gène BRX rend la plante plus résistante aux sols acides. Dans l'optique de mieux comprendre cette valeur adaptative du mutant brx, nous avons analysé dans quels tissus le gène BRX jouait un rôle important. Nous avons pu mettre en évidence que BRX est important pour le développement du protophloème. Le protophloème est un élément du système vasculaire de la plante. En général, les plantes supérieures possèdent deux systèmes de transport à longue distance. L'un d'eux, appelé xylème, transporte l'eau et les nutriments absorbés du sol par les racines vers les feuilles. Les feuilles sont le siège du processus de photosynthèse au cours duquel sont produits des sucres qui devront être distribués partout dans les autres parties de la plante. Le tissu cellulaire chargé de livrer les produits de la photosynthèse, ainsi que les régulateurs de croissance, est le phloème. Ce dernier regroupe le métaphloème et le protophloème. Le protophloème est essentiel pour la livraison des sucres synthétisés ainsi que des signaux de croissance aux pointes des racines, centres organogéniques responsables de la production de nouvelles cellules durant la phase de croissance de la racine. La structure du protophloème peut être décrite comme des tubes continus, vides et résistants, faits de cellules spécialisées qui permettent un transport efficace et rapide. Nous avons montré que dans les mutants brx ces canaux de transports sont discontinus car certaines cellules n'ont pas terminé leur cycle de différenciation. Ces cellules obstruent le conduit ce qui fait que les sucres et les signaux de croissance, comme l'auxine, ne peuvent plus être transportés aux méristèmes. En conséquence, la prolifération de l'activité des méristèmes est compromise, ce qui explique les racines courtes. Au lieu d'être délivré aux méristèmes, l'auxine se concentre en amont des méristèmes où cela provoque l'apparition de nouvelles racines branchées et, très probablement, l'activation des pompes à protons. Sur des sols acides, la concentration en ion H+ est très élevée. Ces ions entrent dans les cellules de la racine par diffusion et perturbent notablement la croissance des racines et de la plante en général. Si les cellules de la racine possédaient des pompes à protons hyperactives, elles seraient capable d'évacuer le surplus d'ions H+ en dehors de la cellule, ce qui leur assurerait de meilleures chances de survie sur sols acides. De fait, le mutant brx est capable d'acidifier le milieu de culture dans lequel il est cultivé plus efficacement que la plante sauvage. Ce mutant est également capable de donner plus de progéniture sur ce type de milieu de croissance que les plantes sauvages. Finalement, nous avons trouvé d'autres mutants brx en milieu naturel poussant sur sols acides, ce qui suggère fortement que la mutation du gène BRX est une des causes de l'adaptation aux sols acides. -- Plants as sessile organisms have developed different mechanisms to cope with the complex environmental conditions in which they live. Adaptation is the process through which traits evolve by natural selection to functionally improve in a given environmental context. An adaptation to the environment is characterized by the genetic changes in the entire populations that have been fixed by natural selection over many generations. BREVIS RADIX (BRX) gene was found through natural Arabidopsis accessions screen and was characterized as a root growth regulator since loss-of-function mutants exhibit arrested post-embryonic primary root growth in addition to a more branched root system. Although brx loss-of-function causes a complete alteration in root architecture, BRX activity is only required in the root vasculature, in particular in protophloem cell file. Protophloem is a part of the phloem transport network and is responsible for delivery of photo-assimilates and growth regulators, coming from the shoot through mature phloem component - metaphloem, to the all plant primary meristems. In order to perform its function, protophloem is the first cell file to differentiate within the root meristem. During this process, protophloem cells undergo a partial programmed cell death, during which they build a thicker cell wall, degrade nucleus and tonoplast while plasma membrane stays functional. Interestingly, protophloem cells enter elongation process only after differentiation into sieve elements is completed. Here we show that brx mutants fail to differentiate protophloem cell file properly, a phenotype that can be distinguished by a presence of a "gap" cells, non-differentiated cells between two flanking differentiated cells. Discontinuity of protophloem differentiation in brx mutants is considered to be a consequence of local hyperactivity of CLAVATA3/EMBRYO SURROUNDING REGION 45 (CLE45) - BARELY ANY MERISTEM 3 (BAM3) signaling module. Interestingly, a CLE45 activity, most probably at the level of receptor binding, can be modulated by apoplastic pH. Altogether, our results imply that the activity of proton pumps, expressed in non-differentiated cells of protophloem, must be maintained under certain threshold, otherwise CLE45-BAM3 signaling pathway will be stimulated and in turn protophloem will not differentiate. Based on vacuolar morphology, a premature cell wall acidification in brx mutants stochastically prevents the protophloem differentiation. Only after protophloem differentiates, proton pumps can be activated in order to acidify apoplast and to support enucleated protophloem multifold elongation driven by surrounding cells growth. Finally, the protophloem differentiation failure would result in an auxin "traffic jam" in the upper parts of the root, created from the phloem-transported auxin that cannot be efficiently delivered to the meristem. Physiologically, auxin "leakage" from the plant vasculature network could have various consequences, since auxin is involved in the regulation of almost every aspect of plant growth and development. Thus, given that auxin stimulates lateral roots initiation and growth, this scenario explains more branched brx root system. Nevertheless, auxin is considered to activate plasma membrane proton pumps. Along with this, it has been shown that brx mutants acidify media much more than the wild type plants do, a trait that was proposed as an adaptive feature of naturally occurring brx null alleles in Arabidopsis populations found on acidic soils. Additionally, in our study we found that most of accessions originally collected from acidic sampling sites exhibit hypersensitivity to CLE45 treatment. This implies that adaptation of plants to acidic soil involves a positive selection pressure against upstream negative regulators of CLE45-BAM3 signaling, such as BRX. Perspective analysis of these accessions would provide more profound understanding of molecular mechanisms underlying plant adaptation to acidic soils. All these results are suggesting that targeting of the factors that affect protophloem differentiation is a good strategy of natural selection to change the root architecture and to develop an adaptation to a certain environment. -- Les plantes comme organismes sessiles ont développé différents mécanismes pour s'adapter aux conditions environnementales complexes dans lesquelles elles vivent. L'adaptation est le processus par lequel des traits vont évoluer via la sélection naturelle vers une amélioration fonctionnelle dans un contexte environnemental donné. Une adaptation à l'environnement est caractérisée par des changements génétiques dans des populations entières qui ont été fixés par la sélection naturelle sur plusieurs générations. Le gène BREVIS RADIX (BRX) a été identifié dans le crible d'une collection d'accessions naturelles d'Arabidopsis et a été caractérisé comme un régulateur de la croissance racinaire étant donné que le mutant perte-de-fonction montre une croissance racinaire primaire arrêtée au stade post-embryonnaire et présente de plus un système racinaire plus ramifié que la plante sauvage. Bien que le mutant perte-de-fonction brx cause une altération complète de l'architecture racinaire, l'activité de BRX n'est requise que dans la vascularisation racinaire, en particulier au niveau du protophloème. Le protophloème est un composant du réseau de transport du phloème et est responsable du transit des dérivés de la photosynthèse ainsi que des régulateurs de croissances, venant de la partie aérienne par le phloème mature (métaphloème) vers tous les méristèmes primaires de la plante. Pour pouvoir réaliser sa fonction, le protophloème est la première file de cellules à se différencier à l'intérieur du méristème de la racine. Pendant ce processus, les cellules du protophloème subissent une mort cellulaire programmée partielle durant laquelle elles épaississent leur paroi cellulaire, dégradent le noyau et le tonoplaste tandis que la membrane plasmique demeure fonctionnelle. De manière intéressante, les cellules du protophloème entament le processus d'allongement seulement après que la différenciation en tubes criblés soit complète. Ce travail montre que le mutant brx est incapable de mener à bien la différenciation de la file de cellules du protophloème, phénotype qui peut être visualisé par la présence de cellules 'trous', de cellules non différenciées entourées de deux cellules différenciées. La discontinuité de la différenciation du phloème dans le mutant brx est considérée comme la conséquence de l'hyperactivité localisée du module de signalisation CLA VA TA3/EMBRYO SURROUNDING REGION 45 (CLE45) - BARELY ANY MERISTEM 3 (BAM3). De manière intéressante, l'activité de CLE45, très probablement au niveau de la liaison avec le récepteur, peut être modulé par le pH apoplastique. Pris ensemble, nos résultats impliquent que l'activité des pompes à protons, actives dans les cellules non différenciées du protophloème, doit être maintenue en dessous d'un certain seuil autrement la cascade de signalisation CLE45-BAM3 serait stimulée, en conséquence de quoi le protophloème ne pourrait se différencier. D'après la morphologie vacuolaire, une acidification prématurée de la paroi cellulaire dans le mutant brx empêche la différenciation du protophloème de manière stochastique. Une fois que le protophloème se différencie, les pompes à protons peuvent alors être activées afin d'acidifier l'apoplaste et ainsi faciliter l'allongement des cellules énuclées du protophloème, entraînées par la croissance des cellules environnantes. Finalement, la différenciation défectueuse du protophloème produit une accumulation d'auxine dans la partie supérieure de la racine car le phloème ne peut plus acheminer efficacement l'auxine au méristème. Physiologiquement, la 'fuite' d'auxine à partir du réseau vasculaire de la plante peut avoir des conséquences variées puisque l'auxine est impliquée dans la régulation de la majorité des aspects de la croissance et développement de la plante. Etant donné que l'auxine stimule l'initiation et développement des racines latérales, ce scénario pourrait expliquer le système racinaire plus ramifié du mutant brx. En plus, l'auxine est considérée comme un activateur des pompes à protons. Par ailleurs, nous avons montré que les mutants brx ont la capacité d'acidifier le milieu plus efficacement que les plantes sauvages, une caractéristique des populations sauvages <¥Arabidopsis poussant sur des sols acides et contenant les allèles délétés brx. De plus, dans nos résultats nous avons mis en évidence que la plupart des accessions collectées originellement sur des sites acidophiles montre une hypersensibilité au traitement par CLE45. Ceci implique que l'adaptation des plantes aux sols acides repose sur la pression de sélection positive à rencontre des régulateurs négatifs de CLE45- BAM3, situés en amont de la cascade, tel le produit du gène BRX. Les analyses de ces accessions pourraient aboutir à une meilleure compréhension des mécanismes moléculaires responsables de l'adaptation des plantes aux sols acides. Tous nos résultats suggèrent que le ciblage des facteurs affectant la différenciation du protophloème serait une stratégie gagnante dans la sélection naturelle pour changer l'architecture de la racine et ainsi s'adapter efficacement à un nouvel environnement.

Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. Methodology/Principal Findings: To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n=38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n=25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9,HOXA9,AHR,NR2F2 ,and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. Conclusions: We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours.

Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells.

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis.

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51's localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8(+) cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.

Degeneracy of antigen recognition as the molecular basis for the high frequency of naive A2/Melan-a peptide multimer(+) CD8(+) T cells in humans.

In contrast with the low frequency of most single epitope reactive T cells in the preimmune repertoire, up to 1 of 1,000 naive CD8(+) T cells from A2(+) individuals specifically bind fluorescent A2/peptide multimers incorporating the A27L analogue of the immunodominant 26-35 peptide from the melanocyte differentiation and melanoma associated antigen Melan-A. This represents the only naive antigen-specific T cell repertoire accessible to direct analysis in humans up to date. To get insight into the molecular basis for the selection and maintenance of such an abundant repertoire, we analyzed the functional diversity of T cells composing this repertoire ex vivo at the clonal level. Surprisingly, we found a significant proportion of multimer(+) clonotypes that failed to recognize both Melan-A analogue and parental peptides in a functional assay but efficiently recognized peptides from proteins of self- or pathogen origin selected for their potential functional cross-reactivity with Melan-A. Consistent with these data, multimers incorporating some of the most frequently recognized peptides specifically stained a proportion of naive CD8(+) T cells similar to that observed with Melan-A multimers. Altogether these results indicate that the high frequency of Melan-A multimer(+) T cells can be explained by the existence of largely cross-reactive subsets of naive CD8(+) T cells displaying multiple specificities.

Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells

BACKGROUND: Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB) is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. RESULTS: We show here that the HDACi Sodium Butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA) strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. CONCLUSION: HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.

Processing of metacaspase into a cytoplasmic catalytic domain mediating cell death in Leishmania major.

Metacaspases are cysteine peptidases that could play a role similar to caspases in the cell death programme of plants, fungi and protozoa. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harbouring a central domain with the catalytic dyad histidine and cysteine as found in caspases. In this study, we investigated the processing sites important for the maturation of LmjMCA catalytic domain, the cellular localization of LmjMCA polypeptides, and the functional role of the catalytic domain in the cell death pathway of Leishmania parasites. Although LmjMCA polypeptide precursor form harbours a functional mitochondrial localization signal (MLS), we determined that LmjMCA polypeptides are mainly localized in the cytoplasm. In stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain. This domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion. These data provide experimental evidences of the importance of LmjMCA processing into an active catalytic domain and of its role in disrupting mitochondria, which could be relevant in the design of new drugs to fight leishmaniasis and likely other protozoan parasitic diseases.

Clinical and molecular characteristics of HNSCC patients with brain metastases: a retrospective study.

Among the metastasis patterns of head and neck squamous cell carcinoma (HNSCC), intracranial spread is a rare but dreaded event. To date only very few cases have been reported and clinical and molecular data are sparse. We screened our archives for HNSCC patients from 1992 to 2005 who were diagnosed with brain metastases (BM). For retrospective analysis, all clinico-pathological data including disease-free survival (DFS), local progression-free survival (LPFS), and overall survival (OS) were compiled. Additionally, we assessed the mutational status of the TP53 gene and the prevalence of HPV serotypes by PCR and Sanger sequencing. Immunohistochemistry was applied to detect p16INK4A expression levels as surrogate marker for HPV infection. The prevalence rate of BM in our cohort comprising 193 patients with advanced HNSCC was 5.7 %. Of 11 patients with BM, 3 were female and 9 were male. Seven of the primary tumors were of oropharyngeal origin (OPSCC). LPFS of the cohort was 11.8 months, DFS was 12.1 months and OS was 36.0 months. After the diagnosis of BM, survival was 10.5 months. Five tumors showed a mutation in the TP53 gene, while five of the seven OPSCC tumors had a positive HPV status displaying infection with serotype 16 in all cases. Compared with patients who harbored TP53wt/HPV-positive tumors, patients with TP53 mutations showed a poor prognosis. Compared with the whole cohort, the interval between diagnosis of the primary and the detection of BM was prolonged in the HPV-infected OPSCC subgroup (26.4 vs. 45.6 months). The prognosis of HNSCC patients with BM is poor. In our cohort, most tumors were OPSCC with the majority being HPV positive. Our study points toward a putatively unusual metastatic behavior of HPV-positive OPSCC.