Understanding the role of CAP proteins in the polarization process of C. elegans

La division cellulaire asymétrique est un processus crucial dans le développement des organismes multicellulaires puisqu’elle permet la génération de la diversité cellulaire. Les cellules qui se divisent de façon asymétrique doivent tout d’abord se polariser et correctement orienter leur fuseau mitotique pour ségréger des déterminants cellulaires en deux entités distinctes. L’embryon du nématode C. elegans est un modèle robuste et largement utilisé pour étudier la division cellulaire asymétrique. Dans cet embryon, le point d'entrée du spermatozoïde détermine l'axe de polarité antéro-postérieur. Suite à la fécondation, le cortex embryonnaire est uniformément contractile et un complexe conservé formé des protéines PAR-3, PAR-6 et PKC-3 (nommé complexe PAR-3 ci-dessous) est localisé sur l'ensemble du cortex. La complétion de la méiose maternelle induit une relaxation corticale au postétieur et un flux cortical vers l’antérieur de l’embryon. Ces contractions corticales asymétriques mènent à la formation d'un domaine antérieur contenant le complexe PAR-3, tandis que le cortex postérieur, dont le complexe PAR-3 s’est délocalisé, est enrichi avec les protéines PAR-2 et PAR-1. Par conséquent, les domaines formés par les protéines PAR définissent un pôle antérieur et un pôle postérieur dans l'embryon suite au remodelage du cytosquelette. Les protéines PAR-4 et PAR-5 restent localisées de façon uniforme dans l'embryon. Curieusement, les protéines PAR exercent une régulation par rétroaction sur la contractilité corticale. Il a été montré qu’une des protéines PAR récemment identifiée, PAR-5, est orthologue à la protéine adaptatrice 14-3-3 et joue un rôle important dans la contractilité corticale. En dépit de son rôle central dans la contractilité corticale et le processus de polarisation cellulaire, le mécanisme par lequel PAR-5 régule la contractilité corticale n’est pas bien compris. Le but de ce projet est de mieux comprendre comment PAR-5 et ses interacteurs contrôlent la régulation des contractions corticales et, de ce fait, la polarité cellulaire. Dans un essai de capture de la protéine GST (GST pull-down), nous avons identifié plusieurs nouveaux interacteurs de PAR-5. Parmi ceux-ci, nous avons trouvé CAP-2 (protéine de coiffage de l'actine), qui a été identifiée dans des éxpériences de capture de 14-3-3 dans trois systèmes modèles différents. CAP-2 est un hétérodimère des protéines CAP, qui sont impliquées dans la régulation de l'actine. Nous avons trouvé que la déplétion des protéines CAP par interférence à l’ARN dans des vers de type sauvage mène à une augmentation létalité embryonnaire, ce qui suggère que ces protéines jouent un rôle important dans le développement embryonnaire. L'imagerie en temps réel d'embryons déplétés pour les protéines CAP montre qu’ils ont une diminution des contractions corticales avec un sillon de pseudoclivage mois stable, suggérant un défaut dans la régulation du cytosquelette d'actine-myosine. Ceci a également été confirmé par la diminution de la vitesse et du nombre de foci de NMY-2::GFP. En outre, ces embryons montrent une légère diminution de la taille du croissant cortical de PAR-2 lors de la phase d’établissement de la polarité. Les embryons déplétés en CAP-2 montrent également un retard dans la progression du cycle cellulaire, mais le lien entre ce phénotype et la régulation des contractions corticales reste à être précisé. La caractérisation des protéines CAP, des régulateurs du remodelage du cytosquelette, permettra d'améliorer notre compréhension des mécanismes qui sous-tendent l'établissement et le maintien de la polarité cellulaire, et donc la division cellulaire asymétrique.

New Interaction Partners For Nek4.1 And Nek4.2 Isoforms: From The Dna Damage Response To Rna Splicing.

Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. In humans there are eleven Neks which are involved in different biological functions besides the cell cycle control. Nek4 is one of the largest members of the Nek family and has been related to the primary cilia formation and in DNA damage response. However, its substrates and interaction partners are still unknown. In an attempt to better understand the role of Nek4, we performed an interactomics study to find new biological processes in which Nek4 is involved. We also described a novel Nek4 isoform which lacks a region of 46 amino acids derived from an insertion of an Alu sequence and showed the interactomics profile of these two Nek4 proteins. Isoform 1 and isoform 2 of Nek4 were expressed in human cells and after an immunoprecipitation followed by mass spectrometry, 474 interacting proteins were identified for isoform 1 and 149 for isoform 2 of Nek4. About 68% of isoform 2 potential interactors (102 proteins) are common between the two Nek4 isoforms. Our results reinforce Nek4 involvement in the DNA damage response, cilia maintenance and microtubule stabilization, and raise the possibility of new functional contexts, including apoptosis signaling, stress response, translation, protein quality control and, most intriguingly, RNA splicing. We show for the first time an unexpected difference between both Nek4 isoforms in RNA splicing control. Among the interacting partners, we found important proteins such as ANT3, Whirlin, PCNA, 14-3-3ε, SRSF1, SRSF2, SRPK1 and hNRNPs proteins. This study provides new insights into Nek4 functions, identifying new interaction partners and further suggests an interesting difference between isoform 1 and isoform 2 of this kinase. Nek4 isoform 1 may have similar roles compared to other Neks and these roles are not all preserved in isoform 2. Besides, in some processes, both isoforms showed opposite effects, indicating a possible fine controlled regulation.

Differential gene expression profiling in mucus glands of honey bee (Apis mellifera) drones during sexual maturation

The mating sign that each drone leaves when mating with a queen essentially consists of mucus gland proteins. We employed a Representational Difference Analysis (RDA) methodology to identify genes that are differentially expressed in mucus glands during sexual maturation of drones. The RDA library for mucus glands of newly emerged drones was more complex than that of 8 day-old drones, with matches to 20 predicted genes. Another 26 reads matched to the Apis genome but not to any predicted gene. Since these ESTs were located within ORFs they may represent novel honey bee genes, possibly fast evolving mucus gland proteins. In the RDA library for mucus glands of 8 day-old drones, most reads corresponded to a capsid protein of deformed wing virus, indicating high viral loads in these glands. The expression of two genes encoding venom allergens, acid phosphatase-1 and hyaluronidase, in drone mucus glands argues for their homology with the female venom glands, both associated with the reproductive system.

Transcript profiling of papaya fruit reveals differentially expressed genes associated with fruit ripening

Papaya (Carica papaya L) fruit has a short shelf life due to fast ripening induced by ethylene, but little is known about the genetic control of ripening and attributes of fruit quality. Therefore, we identified ripening-related genes affected by ethylene using cDNA-AFLP (Amplified Fragment Length Polymorphism of cDNA). Transcript profiling of non-induced and ethylene-induced fruit samples was performed, and 71 differentially expressed genes were identified. Among those genes some involved in ethylene biosynthesis, regulation of transcription, and stress responses or plant defence were found (heat shock proteins, polygalacturonase-inhibiting protein, and acyl-CoA oxidases). Several transcription factors were isolated, and except for a 14-3-3 protein, an AP2 domain-containing factor, a salt-tolerant zinc finger protein, and a suppressor of PhyA-105 1, most of them were negatively affected by ethylene, including fragments of transcripts similar to VRN1, and ethylene responsive factors (ERF). With respect to fruit quality, genes related to cell wall structure or metabolism, volatiles or pigment precursors, and vitamin biosynthesis were also found. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Activation of beta(2)-adrenergic receptors hastens relaxation and mediates phosphorylation of phospholamban, troponin I, and C-protein in ventricular myocardium from patients with terminal heart failure

Background-Catecholamines hasten cardiac relaxation through beta-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of beta(1)- and beta(2)-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. Methods and Results-Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 mu mol/L) or zinterol (10 mu mol/L), mediated through beta(2)-adrenergic receptors, and of norepinephrine (10 mu mol/L), mediated through beta(1)-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17, Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15+/-3%, 5+/-2%, and 20+/-3%, respectively, and reduced the time to half-relaxation by 26+/-3%, 21+/-3%, and 37+/-3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14+/-3, 12+/-4, and 12+/-3, troponin I 40+/-7, 33+/-7, and 31+/-6; and C-protein 7.2+/-1.9, 9.3 +/- 1.4, and 7.5 +/- 2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both beta(1)- and beta(2)-adrenergic receptors. Conclusions-Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both beta(1)- and beta(2)-adrenergic receptors, thereby potentially improving diastolic function.

Proteomics of breast cancer for marker discovery and signal pathway profiling

Breast cancer is the most common form of cancer among women and the identification of markers to discriminate tumorigenic from normal cells, as well as the different stages of this pathology, is of critical importance. Two-dimensional electrophoresis has been used before for studying breast cancer, but the progressive completion of human genomic sequencing and the introduction of mass spectrometry, combined with advanced bioinformatics for protein identification, have considerably increased the possibilities for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified markers of potential clinical interest (such as the molecular chaperone 14-3-3 sigma) and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer. As illustrated with fibroblast growth factor-2, a mitogen and motogen factor for breast cancer cells, proteomics is a powerful approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth. Together with genomics, proteomics is well on the way to molecularly characterizing the different types of breast tumor, and thus defining new therapeutic targets for future treatment.

Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children

The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public) in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters) were analyzed, according to their homogeneous (socioeconomic category and/or same school) and heterogeneous (distinct socioeconomic categories and/or schools) characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0) among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low). Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public) also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a restricted dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < S D < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.

Signal transduction pathways involving the hypertension-related WNK1 and WNK4 protein kinases

Dissertação apresentada para obtenção do Grau de Doutor em Biologia, na especialidade de Genética Molecular, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Taeniosis-cysticercosis complex in individuals of a peasants' settlement (Teodoro Sampaio, Pontal of Paranapanema, SP, Brazil)

In order to evaluate the taeniosis-cysticercosis complex in a population of a peasants' settlement, located at Teodoro Sampaio, state of São Paulo, Brazil (longitude 52° 36'12 ", latitude 22° 17'12 ") a series of laboratory markers were determined. After signing an informed consent, participants answered a standardized questionnaire. To determine anti-Taenia solium cysticercus antibodies, the samples were tested by enzyme linked immunoabsorbent assay using 18-and 14-kDa antigen proteins from vesicular fluid of Taenia crassiceps (VF-Tcra). The reactive and inconclusive ELISA samples were tested by immunoblotting. Total IgE levels were determined by chemmiluminescence's assay and hemogram by flow cytometer flux counter. A total of 84 individuals, 5.9% presented anti-T. solium cysticercus antibodies in ELISA and 3.6% were strongly reactive in the 18/14 kDa immunoblotting confirmatory test. All of the individuals with positive antibodies showed elevated Total IgE levels. We conclude that the frequency of anti-T. solium cysticercus antibodies in this population is higher than other regions considered endemic in São Paulo. Thus, it is important to carry out surveys in Peasants' settlement areas with the objective of establishing public health measures for prevention and control of infectious diseases such as taeniosis-cysticercosis.

Adiposity, hormone replacement therapy use and breast cancer risk by age and hormone receptor status: a large prospective cohort study.

INTRODUCTION Associations of hormone-receptor positive breast cancer with excess adiposity are reasonably well characterized; however, uncertainty remains regarding the association of body mass index (BMI) with hormone-receptor negative malignancies, and possible interactions by hormone replacement therapy (HRT) use. METHODS Within the European EPIC cohort, Cox proportional hazards models were used to describe the relationship of BMI, waist and hip circumferences with risk of estrogen-receptor (ER) negative and progesterone-receptor (PR) negative (n = 1,021) and ER+PR+ (n = 3,586) breast tumors within five-year age bands. Among postmenopausal women, the joint effects of BMI and HRT use were analyzed. RESULTS For risk of ER-PR- tumors, there was no association of BMI across the age bands. However, when analyses were restricted to postmenopausal HRT never users, a positive risk association with BMI (third versus first tertile HR = 1.47 (1.01 to 2.15)) was observed. BMI was inversely associated with ER+PR+ tumors among women aged ≤49 years (per 5 kg/m2 increase, HR = 0.79 (95%CI 0.68 to 0.91)), and positively associated with risk among women ≥65 years (HR = 1.25 (1.16 to 1.34)). Adjusting for BMI, waist and hip circumferences showed no further associations with risks of breast cancer subtypes. Current use of HRT was significantly associated with an increased risk of receptor-negative (HRT current use compared to HRT never use HR: 1.30 (1.05 to 1.62)) and positive tumors (HR: 1.74 (1.56 to 1.95)), although this risk increase was weaker for ER-PR- disease (Phet = 0.035). The association of HRT was significantly stronger in the leaner women (BMI ≤22.5 kg/m2) than for more overweight women (BMI ≥25.9 kg/m2) for, both, ER-PR- (HR: 1.74 (1.15 to 2.63)) and ER+PR+ (HR: 2.33 (1.84 to 2.92)) breast cancer and was not restricted to any particular HRT regime. CONCLUSIONS An elevated BMI may be positively associated with risk of ER-PR- tumors among postmenopausal women who never used HRT. Furthermore, postmenopausal HRT users were at an increased risk of ER-PR- as well as ER+PR+ tumors, especially among leaner women. For hormone-receptor positive tumors, but not for hormone-receptor negative tumors, our study confirms an inverse association of risk with BMI among young women of premenopausal age. Our data provide evidence for a possible role of sex hormones in the etiology of hormone-receptor negative tumors.

Clinicopathologic correlations of renal microthrombosis and inflammatory markers in proliferative lupus nephritis.

INTRODUCTION Microthrombosis is often observed in lupus nephritis (LN) lesions, but its clinical significance is unknown. We evaluated the clinicopathologic correlations of renal microthrombosis and inflammatory markers in LN. METHODS Kidney biopsies from 58 patients with systemic lupus erythematosus (SLE) proliferative nephritis were analyzed with immunohistochemistry (IHC) for intravascular platelet aggregates (CD61), macrophagic infiltration (CD68), and activated complement deposition (C4d). Clinical data at the time of kidney biopsy and follow-up were analyzed with regard to pathologic IHC data. RESULTS Microthrombosis was present in 52% of the tissues. It was significantly more prevalent in patients with antiphospholipid antibodies (aPLs) (62% versus 42%). The presence of microthrombosis significantly correlated with higher macrophagic infiltration. Macrophagic infiltration but not microthrombosis was significantly correlated with C4d deposition. Only macrophagic infiltration showed a correlation with SLE and renal activity (proteinuria and active sediment), whereas neither the presence of CD61+ microthrombi nor the extent of C4d deposition correlated with LN severity or outcome. CONCLUSIONS Microthrombosis is associated with higher macrophagic infiltration in LN but does not seem to increase independently the severity of renal damage. Macrophagic infiltration was the best marker of SLE and renal activity in this LN series.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

Principio de unidad familiar y cambio de vecindad civil por residencia. Comentario a la STS, 1ª, 14.9.2009

En la línea apuntada por la mayoría de la doctrina, la STS de 14.9.2009 considera que desde la entrada en vigor de la Constitución española (1978) debía entenderse derogada la regla de propagación a la esposa de la vecindad civil del marido prevista en el art. 14.4 CCe [redacción de 1974]; ello supone que desde entonces la mujer casada pudo cambiar autónomamente de vecindad civil. Partiendo de ello, en el caso enjuiciado, la mujer cambió de vecindad civil por residencia continuada en Cataluña durante más de diez años sin declaración en contrario (art. 14.3.2.o CCe [1974] y 14.5.2.o CCe [1990]), vecindad civil catalana que mantenía en el momento de su fallecimiento, pese a haber realizado distintas manifestaciones ¿que resultan ineficaces? en el sentido de ostentar o de querer conservar la vecindad civil navarra.

Principi d'unitat familiar i canvi de veïnatge civil per residència. Comentari a la STS, 1a, 14.9.2009

En la línia del que havia apuntat la majoria de la doctrina, la STS de 14.9.2009 considera que des de l¿entrada en vigor de la Constitució espanyola (1978) havia d'entendre's derogada la regla de propagació a l¿esposa del veïnatge civil del marit prevista a l¿art. 14.4 Codi civil espanyol [redacció de 1974]; això suposa que des d¿aleshores la dona casada podia canviar autònomament de veïnatge civil. Partint d'això, en el cas judicat, la dona va canviar de veïnatge civil per residència continuada a Catalunya durant més de deu anys sense declaració en contra (art. 14.3.2n CCe [1974] i 14.5.2n CCe [1990]), veïnatge civil català que mantenia en el moment de la seva mort, malgrat haver realitzat diferents manifestacions -que resulten ineficaces- en el sentit de tenir o de voler conservar el veïnatge civil navarrès.