Analysen der beiden cis-regulierenden Sequenzelemente translational control element (TCE) und cytoplasmic polyadenylation element (CPE) in der Drosophila-Spermatogenese

Ein essentieller Bestandteil in dem Mechanismus der Translationskontrolle sind RNA-Pro­tein-Wechselwirkungen. Solche Interaktionen konnten in Translationssystemen an zwei unabhängigen cis-regulierenden Elementen durch in vitro-Bindungsanalysen mit individu­ellen rekombinanten Proteinen dokumentiert werden. Im Fall des translational control elements (TCE), welches ein konserviertes Sequenz-Ele­ment in der Mst(3)CGP-Genfamilie darstellt, wird eine negative Translationskontrolle durch die Bindung der Proteine CG3213, CG12470, CG1898, dFMR1, Exuperantia und Orb2 an diese Sequenz vermittelt (Stinski, 2011). Neben den in Bindungsstudien positiv getesteten Kandidaten dFMR1 und Orb2 (Stinski, 2011) wurde in der vorliegenden Dis­sertation CG3213 als weiterer direkter Bindungspartner an das TCE dokumentiert. Ein Abgleich der genomweiten Zusammenstellung von Proteininteraktionen in der Datenbank InterologFinder lieferte zwei weitere potentielle Kandidaten: CG34404 und CG3727. Al­lerdings schließen Northern-Analysen und das Proteinexpressionsmuster eine zentrale Rolle in der Drosophila-Spermatogenese für diese nahezu aus. In Kolokalisationsstudien einiger TCE-Komplex-Kandidaten mit CG3213 als Referenz konnten eindeutige Überein­stimmungen der Fluoreszenzmuster mit CG12470 in der postmeiotischen Phase be­schrieben werden, wohingegen mit Orb2 (postmeiotisch) und CG1898 (prämeiotisch) nur eine geringe Kolokalisation erkannt wurde. Punktstrukturen in den Verteilungsmustern sowohl von CG3213 als auch von CG12470 ließen sich nicht mit ER- und mitochondrien­spezifischen Markern korrelieren. Im Anschluss der Meiose konnte eine deutliche Intensitätserhöhung des CG3213-Proteins beobachtet werden, was eventuell durch eine veränderte Translationseffizienz zustande kommen könnte. Exuperantia (Exu) stellt einen bekannten Regulator für eine Reihe von translationskontrollierten mRNAs dar (Wang und Hazelrigg, 1994). Die Quantifizierungen der CG3213-mRNA in exu-mutantem Hintergrund bestätigen, dass auch die Transkript­menge der CG3213-mRNA durch Exu reguliert wird, was die obige Interpretation stützen würde. Für das zweite cis-regulierende Element, das cytoplasmic polyadenylation element (CPE), konnte eine direkte Bindung mit dem CPEB-Homolog in Drosophila (Orb2) gezeigt wer­den, welches auch eine Komponente des mst87F-RNP-Komplexes ist. Ein vermuteter Interaktionspartner dieses CPEBs ist Tob, weshalb die Verteilung beider Proteine in einem Kombinationsstamm verglichen wurde. In dem teilweise übereinstimmenden Fluoreszenz­muster ist Tob an den distalen Spermatidenenden auffallend konzentriert. Das gesamte Tob-Muster jedoch legt eine Verteilung in den Mitochondrien nahe, wie die MitoTracker®-Färbung belegt. Somit wurde erstmals ein Mitglied der Tob/BTG-Genfamilie in der Droso­phila-Spermatogenese mit Mitochondrien in Verbindung gebracht. Die Lokalisierung die­ser Proteine ist bislang unklar, jedoch konnte eine Kernlokalisation trotz der N-terminalen NLS-Sequenz mit Hilfe einer Kernfärbung ausgeschlossen werden.

Selecting Relevant Genes with a Spectral Approach

Array technologies have made it possible to record simultaneously the expression pattern of thousands of genes. A fundamental problem in the analysis of gene expression data is the identification of highly relevant genes that either discriminate between phenotypic labels or are important with respect to the cellular process studied in the experiment: for example cell cycle or heat shock in yeast experiments, chemical or genetic perturbations of mammalian cell lines, and genes involved in class discovery for human tumors. In this paper we focus on the task of unsupervised gene selection. The problem of selecting a small subset of genes is particularly challenging as the datasets involved are typically characterized by a very small sample size ?? the order of few tens of tissue samples ??d by a very large feature space as the number of genes tend to be in the high thousands. We propose a model independent approach which scores candidate gene selections using spectral properties of the candidate affinity matrix. The algorithm is very straightforward to implement yet contains a number of remarkable properties which guarantee consistent sparse selections. To illustrate the value of our approach we applied our algorithm on five different datasets. The first consists of time course data from four well studied Hematopoietic cell lines (HL-60, Jurkat, NB4, and U937). The other four datasets include three well studied treatment outcomes (large cell lymphoma, childhood medulloblastomas, breast tumors) and one unpublished dataset (lymph status). We compared our approach both with other unsupervised methods (SOM,PCA,GS) and with supervised methods (SNR,RMB,RFE). The results clearly show that our approach considerably outperforms all the other unsupervised approaches in our study, is competitive with supervised methods and in some case even outperforms supervised approaches.

Dual-specificity phosphatases in the hypo-osmotic stress response of keratin-defective epithelial cell lines

Although mutations in intermediate filament proteins cause many human disorders, the detailed pathogenic mechanisms and the way these mutations affect cell metabolism are unclear. In this study, selected keratin mutations were analysed for their effect on the epidermal stress response. Expression profiles of two keratin-mutant cell lines from epidermolysis bullosa simplex patients (one severe and one mild) were compared to a control keratinocyte line before and after challenge with hypo-osmotic shock, a common physiological stress that transiently distorts cell shape. Fewer changes in gene expression were found in cells with the severely disruptive mutation (55 genes altered) than with the mild mutation (174 genes) or the wild type cells (261 genes) possibly due to stress response pre-activation in these cells. We identified 16 immediate-early genes contributing to a general cell response to hypo-osmotic shock, and 20 genes with an altered expression pattern in the mutant keratin lines only. A number of dual-specificity phosphatases (MKP-1, MKP-2, MKP-3, MKP-5 and hVH3) are differentially regulated in these cells, and their downstream targets p-ERK and p-p38 are significantly up-regulated in the mutant keratin lines. Our findings strengthen the case for the expression of mutant keratin proteins inducing physiological stress, and this intrinsic stress may affect the cell responses to secondary stresses in patients' skin.

The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat ( RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT ( HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 ( AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract - but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.

Clustered Fox genes in lophotrochozoans and the evolution of the bilaterian Fox gene cluster

FoxC, FoxF, FoxL1 and FoxQ1 genes have been shown to be clustered in some animal genomes, with mesendodermal expression hypothesised as a selective force maintaining cluster integrity. Hypotheses are, however, constrained by a lack of data from the Lophotrochozoa. Here we characterise members of the FoxC, FoxF, FoxL1 and FoxQ1 families from the annelid Capitella teleta and the molluscs Lottia gigantea and Patella vulgata. We cloned FoxC, FoxF, FoxL1 and FoxQ1 genes from C. teleta, and FoxC, FoxF and FoxL1 genes from P. vulgata, and established their expression during development. We also examined their genomic organisation in C. teleta and L. gigantea, and investigated local syntenic relationships. Our results show mesodermal and anterior gut expression is a common feature of these genes in lophotrochozoans. In L. gigantea FoxC, FoxF and FoxL1 are closely linked, while in C. teleta Ct-foxC and Ct-foxL1 are closely linked, with Ct-foxF and Ct-foxQ1 on different scaffolds. Adjacent to these genes there is limited evidence of local synteny. This demonstrates conservation of genomic organisation and expression of these genes can be traced in all three bilaterian Superphyla. These data are evaluated against competing theories for the long-term maintenance of gene clusters.

Clock Genes, Melanopsins, Melatonin, and Dopamine Key Enzymes and Their Modulation by Light and Glutamate in Chicken Embryonic Retinal Cells

The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 mu M glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 mu M glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli. (Author correspondence: amdlcast@ib.usp.br).

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Characterization of Paracoccidioides brasiliensis COX9, COX12, and COX16 respiratory genes

Paracoccidioides brasiliensis is a thermo-dimorphic fungus that is the causative agent of paracoccidioidomyicosis (PCM), a human systemic granulomatous mycosis found in Latin America. Dimorphic transition from mycelium to yeast is required for establishing pathogenicity. Dimorphism is marked by changes in mitochondrial physiology, including modulation of respiration rate. In this work, we present the identification of three P. brasiliensis nuclear genes PbCOX9, PbCOX12, and PbCOX16 that code for structural sub-units and a putative assembly facilitator (PbCOX16) of the mitochondrial cytochrome c oxidase (COX), the terminal enzyme complex of the respiratory chain. We measured their expression pattern during the dimorphic transition from mycelium to yeast and back by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). Our results show that messages from these genes increase during the mycelium to yeast transition and decrease during the opposite conversion. This result supports active mitochondrial participation in the transition. Heterologous complementation of the corresponding Saccharomyces cerevisiae null mutant with the PbCOX9 gene was successfully obtained. (C) 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Caracterização e comparação de genes expressos em ápices meristemáticos de cana-de-açúcar cultivados em SP e RN

In this work, we used sugarcane as a model due to its importance for sugar and ethanol production. Unlike the current plant models, sugarcane presents a complex genetics and an enormous allelic variation. Here, we report the analysis of SAGE libraries produced using the shoot apical meristem from contrasted genotypes by flowering induction (non-flowering vs. early-flowering varieties) grown under São Paulo state conditions. The expression pattern was analyzed using samples from São Paulo (SP) and Rio Grande do Norte (RN) states. These results showed that cDNAs identified by SAGE libraries had differential expression only in São Paulo state samples. Furthermore, the cDNA identified CYP (Citocrome P450) was chosen for in silico and genome characterization because it was found in SAGE libraries and subtractive libraries from samples from RN. Phylogenetic trees showed the relationship for these sequences. Furthermore, the qRT-PCR for CYP showed a potential role as flowering indutor for RN samples considering different isophorms. Considering the results present here, it can be consider that CYP gene may be used as molecular marker

Influência do sistema anteoxidante da catalase no ciclo do glioxilato durante o estabelecimento pós-germinativo de cártamo (Carthamus tinctorius L.) e de girassol (Helianthus annuus L.)

Oilseeds are a high-value natural resource, due to its use as a substitute for petroleum. However, the storage time can reduce seed viability and oil quality. Therefore, scientific efforts have been made to provide a increment of storage time, germination rates and plant establishment of high-value oilseeds. The seedling establishment depends of the plant pass over the functional transition stage, characterized by a metabolic change from heterotrophic condition to autotrophic one. The storage oil mobilization is performed by β-oxidation process and the glyoxylate cycle. Also, the functional transition involves acclimation to photosynthetic condition, which generally includes the participation of antioxidant system and the reactive oxygen species, the latter are produced in various reactions of primary and secondary metabolism. In the present study, Catalase was inhibited during the functional transition of sunflower and safflower, after were performed many analyzes to elucidate the effects caused on the SOD and APX antioxidant systems. Also, were checked the changes in expression pattern of the glyoxylate cycle enzymes markers, ICL and MLS. It was observed that after CAT inhibition, the SOD and APX antioxidant systems allow the seedling establishment. Besides, was verified that both oilseeds can be accelerate the reverse mobilization and the photosynthetic establishment when Catalase activity has dramatically decreased

Emprego da citometria de fluxo na avaliação do perfil imunofenotípico de pacientes com leucemia linfocítica crônica

Chronic lymphocytic leukemia (B-CLL) is a clonal proliferation of mature B lymphocytes characterized by indolent clinical course. Biologically this clonallity is characterized by low expression of surface immunoglobulin (sIg) with restriction to a single immunoglobulin light chain associated with high expression of CD5 antigen and positivity to B cell antigens lymphocytes such as CD19, CD20 and CD23 and negativity to FMC7. The immunological profile and morphological analysis of lymphoid cells are the main means for the differential diagnosis of B-CLL from other chronic lymphoproliferative diseases. The aim of this study was to evaluate the expression pattern of a variety of membrane antigens in leukemic cells originating from patients with B-CLL. In this study, peripheral blood samples from 80 patients with B-CLL were analyzed by multiparametric flow cytometry in addition to routine hematologic exams, using a panel of monoclonal antibodies (MoAb): CD45/CD14, CD3/CD19/CD45, CD4/CD8 / CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b and also IgM/gD, kappa and lambda immunoglobulin light chains for the detection of surface immunoglobulin and clonal restriction for immunoglobulin light chain. The Hematological data were obtained from the hematological analyzer and cytomorphological analysis in blood film stained by Leishmann. The study samples consisted of 45 men and 35 women, ages ranging from 55 to 84 years (mean 65 years). Complete white blood count showed count ranging from 10.0 to 42.0 x 109/l. (mean 50.0 x 109/l) and lymphocytes count greater than 5.0 x 109/l in all cases. The neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/CD20+/HLADR+/CD23+) in the vast majority of the cases, associated to failed to stain for T cell markers (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 and FMC7. Leukemic cells of most patients also expressed low intensity of IgM and IgD with restricted kappa light chain, in most cases (59,7%). This observation highlights the importance of immunophenotyping for correct diagnosis of chronic lymphoproliferative syndromes and the panel of MoAb used was sufficient for diagnostic confirmation of B-CLL

Cortactin is associated with perineural invasion in the deep invasive front area of laryngeal carcinomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chicken skeletal muscle-associated macroarray for gene discovery

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Histological Characterization and Immunohistochemical Localization of Ki-67 and VEGF Factors in Bitches' Corpus Luteum at Cyclic and Early and Late Gestational Diestrous

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular weight estimation of esterase isoenzymes in closely related Drosophila Species (Diptera: Drosophilidae) in non-denaturing polyacrylamide gel electrophoresis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)