Characterization of monocarboxylate transporters (MCTs) expression in soft tissue sarcomas: distinct prognostic impact of MCT1 sub-cellular localization

Background: Soft tissue sarcomas (STSs) are a group of neoplasms, which, despite current therapeutic advances, still confer a poor outcome to half of the patients. As other solid tumors, STSs exhibit high glucose consumption rates, associated with worse prognosis and therapeutic response. As highly glycolytic tumors, we hypothesized that sarcomas should present an increased expression of lactate transporters (MCTs).Methods: Immunohistochemical expression of MCT1, MCT2, MCT4 and CD147 was assessed in a series of 86 STSs and the expression profiles were associated with patients' clinical-pathological parameters.Results: MCT1, MCT4 and CD147 were mainly observed in the plasma membrane of cancer cells (around 60% for MCTs and 40% for CD147), while MCT2 was conspicuously found in the cytoplasm (94.2%). Importantly, we observed MCT1 nuclear expression (32.6%). MCT1 and MCT4, alone or co-expressed with CD147 in the plasma membrane, were associated with poor prognostic variables including high tumor grade, disease progression and shorter overall survival. Conversely, we found MCT1 nuclear expression to be associated with low grade tumors and longer overall survival.Conclusions: The present work represents the first report of MCTs characterization in STSs. We showed the original finding of MCT1 expression in the nucleus. Importantly, opposite biological roles should be behind the dual sub-cellular localization of MCT1, as plasma membrane expression of MCT1 is associated with worse patients' prognosis, while nuclear expression is associated with better prognosis.

Fast determination of beef quality parameters with time-domain nuclear magnetic resonance spectroscopy and chemometrics

The noteworthy of this study is to predict seven quality parameters for beef samples using time-domain nuclear magnetic resonance (TD-NMR) relaxometry data and multivariate models. Samples from 61 Bonsmara heifers were separated into five groups based on genetic (breeding composition) and feed system (grain and grass feed). Seven sample parameters were analyzed by reference methods; among them, three sensorial parameters, flavor, juiciness and tenderness and four physicochemical parameters, cooking loss, fat and moisture content and instrumental tenderness using Warner Bratzler shear force (WBSF). The raw beef samples of the same animals were analyzed by TD-NMR relaxometry using Carr-Purcell-Meiboom-Gill (CPMG) and Continuous Wave-Free Precession (CWFP) sequences. Regression models computed by partial least squares (PLS) chemometric technique using CPMG and CWFP data and the results of the classical analysis were constructed. The results allowed for the prediction of aforementioned seven properties. The predictive ability of the method was evaluated using the root mean square error (RMSE) for the calibration (RMSEC) and validation (RMSEP) data sets. The reference and predicted values showed no significant differences at a 95% confidence level.

Análise da relação filogenética entre Triatoma carcavalloi Jurberg, Rocha; Lent, 1998; Triatoma circummaculata Stal, 1859; Triatoma klugi Carcavallo, Jurberg, Lent; Galvão, 2001 e Triatoma rubrovaria Blanchard, 1843 (Hemiptera, Reduviidae) baseada no sequenciamento de genes do DNA mitocondrial e nuclear

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

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Microsatellite in Aeschynomene falcata (Leguminosae): diversity, cross-amplification, and chromosome localization

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

ITScan: a web-based analysis tool for Internal Transcribed Spacer (ITS) sequences

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

Phylogeny of Lundia (Bignoniaceae) based on ndhF and PepC sequences

The circumscription of genera belonging to tribe Bignonieae (Bignoniaceae) has traditionally been complex, with only a few genera having stable circumscriptions in the various classification systems proposed for the tribe. The genus Lundia, for instance, is well characterized by a series of morphological synapomorphies and its circumscription has remained quite stable throughout its history. Despite the stable circumscription of Lundia, the circumscription of species within the genus has remained problematic. This study aims to reconstruct the phylogeny of Lundia in order to refine species circumscriptions, gain a better understanding of relationships between taxa, and identify potential morphological synapomorphies for species and major clades. We sampled 26 accessions representing 13 species of Lundia, and 5 outgroups, and reconstructed the phylogeny of the genus using a chloroplast (ndhF) and a nuclear marker (PepC). Data derived from sequences of the individual loci were analyzed using parsimony and Bayesian inference, and the combined molecular dataset was analyzed with Bayesian methods. The monophyly of Lundia nitidula, a species with a particularly complex circumscription, was tested using Shimodaira-Hasegawa (SH) test and the approximately unbiased test for phylogenetic tree selection (AU test). In addition, 40 morphological characters were mapped onto the tree that resulted from the analysis of the combined molecular dataset in order to identify morphological synapomorphies of individual species and major clades. Lundia and most species currently recognized within the genus were strongly supported as monophyletic in all analyses. One species, Lundia nitidula, was not resolved as monophyletic, but the monophyly of this species was not rejected by the AU and SH tests. Lundia sect. Eriolundia is resolved as paraphyletic in all analyses, while Lundia sect. Eulundia is monophyletic and supported by the same morphological characters traditionally used to circumscribe this section. The phylogeny of Lundia contributed important information for a better circumscription of species and served as basis the taxonomic revision of the genus.

Genetic variability in mitochondrial and nuclear genes of Larus dominicanus (Charadriiformes, Laridae) from the Brazilian coast

Several phylogeographic studies of seabirds have documented low genetic diversity that has been attributed to bottleneck events or individual capacity for dispersal. Few studies have been done in seabirds on the Brazilian coast and all have shown low genetic differentiation on a wide geographic scale. The Kelp Gull is a common species with a wide distribution in the Southern Hemisphere. In this study, we used mitochondrial and nuclear markers to examine the genetic variability of Kelp Gull populations on the Brazilian coast and compared this variability with that of sub-Antarctic island populations of this species. Kelp Gulls showed extremely low genetic variability for nnitochondrial markers (cytb and ATPase) and high diversity for a nuclear locus (intron 7 of the beta-fibrinogen). The intraspecific evolutionary history of Kelp Gulls showed that the variability found in intron 7 of the beta-fibrinogen gene was compatible with the variability expected under neutral evolution but suggested an increase in population size during the last 10,000 years. However, none of the markers revealed evidence of a bottleneck population. These findings indicate that the recent origin of Kelp Gulls is the main explanation for their nuclear diversity, although selective pressure on the mtDNA of this species cannot be discarded.

E-cadherin in canine mast cell tumors: Decreased expression and altered subcellular localization in Grade 3 tumors

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior. (C) 2012 Published by Elsevier Ltd.

Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans

It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of Chironomus tentans provide a suitable model system for studying gene expression in situ, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in C. tentans named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of C. tentans salivary gland cells. Hrp59 is a C. tentans homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in Drosophila cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences. Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1. Hrp65 binds to HEL/UAP56 in C. tentans cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.

LRP1 modulates APP trafficking and APP metabolism within compartments of the secretory pathway. Increased AICD generation is ineffective in nuclear translocation and transcriptional activation

LRP1 modulates APP trafficking and metabolism within compartments of the secretory pathway The amyloid precursor protein (APP) is the parent protein to the amyloid beta peptide (Abeta) and is a central player in Alzheimer’s disease (AD) pathology. Abeta liberation depends on APP cleavage by beta- and gamma-secretases. To date, only a unilateral view of APP processing exists, excluding other proteins, which might be transported together and/or processed dependent on each other by the secretases described above. The low density lipoprotein receptor related protein 1 (LRP1) was shown to function as such a mediator of APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. Therefore, we wanted to investigate whether LRP1 can mediate APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, we demonstrate that APP trafficking is strongly influenced by LRP1 transport through the endoplasmic reticulum (ER) and Golgi compartments. LRP1-constructs with ER- and Golgi-retention motifs (LRP-CT KKAA, LRP-CT KKFF) had the capacity to retard APP trafficking at the respective steps in the secretory pathway. Here, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases. Increased AICD generation is ineffective in nuclear translocation and transcriptional activity A sequence of amyloid precursor protein (APP) cleavages gives rise to the APP intracellular domain (AICD) together with amyloid beta peptide (Abeta) and/or p3 fragment. One of the environmental factors identified favouring the accumulation of AICD appears to be a rise in intracellular pH. This accumulation is a result of an abrogated cleavage event and does not extend to other secretase substrates. AICD can activate the transcription of artificially expressed constructs and many downstream gene targets have been discussed. Here we further identified the metabolism and subcellular localization of the constructs used in this well documented gene reporter assay. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely that cleaved from C83. Furthermore, the AICD surplus is not transcriptionally active but rather remains membrane tethered and free in the cytosol where it interacts with Fe65. However, Fe65 is still essential in AICD mediated transcriptional transactivation although its exact role in this set of events is unclear.

Regelmechanismen und Determinanten der Transkription in Polytänchromosomen von Chironomus und Drosophila

Die kumulative Habil.‐Schrift gründet sich auf 6 Originalpublikationen, die beschreiben: [Sass, H. (1982), Cell 28: 269‐278]. RNA polymerase B in polytene chromosomes: Immunofluorescent and autoradiographic analysis during stimulated and repressed RNA synthesis. Elektronenmikroskopie charakterisierte das C. tentans Balbianiring BR2‐Gen von Speicheldrüsenchromosomen als hoch aktives 5‐6 μm langes single‐copy Gen, das 33/μm RNAPolymerasen B (Pol II) transkribieren (Diss., Sass, H., 1978, Univ. Tübingen). Diese Immunfluoreszenzstudie ortet Pol II in allen Interbanden von Region IV‐3B10‐3B5 des nichtinduzierten BR2. Prominente Fluoreszenz im BR2‐Genort 3B9/10 zeigt, das BR2‐Gen ist präaktiv, wie erwartet. 3H‐Autoradiogramme beweisen, in allen fluoreszierenden BR2, BR1, BR3, Puffs, aufgelockerten Banden, Interbanden und Loci ohne Puffing, synthetisiert Pol II RNA. Die genomweite ständige Pol II‐Präsenz zeigt, dass, wie beim nichtinduzierten BR2‐Gen, bereits schon gebundene Pol II wohl auch andere Gene präaktiviert. So erfolgt die Regulation der Transkription mehr über die transkriptionelle Elongation. Auch durch α‐Amanitin, oder Actinomycin D, oder Hitzeschock in vivo kollabierte BR2, BR1, BR3 besitzen Pol II. [Sass, H. (1984), Chromosoma 90: 20‐25]. Gene identification in polytene chromosomes: some Balbiani ring 2 gene sequences are located in an interband‐like region of Chironomus tentans. Immunfluoreszenz und 3H‐Autoradiographie zeigen, dass Injektionen von DRB in Larven die Balbianiringe (BR) sowie andere Puffs und deren Pol II‐Konzentration dramatisch reduzieren. Trotzdem zeigen 3H‐Uridin markierte Speicheldrüsenchromosomen, dass RNA‐Synthese doch in nichtinduzierten BR2, BR1, BR3 erfolgt, aber nur auf reduziertem Level. Das widerspricht der von Egyházi E. (1975, PNAS 73:947‐950) propagierten „Inhibition of Balbiani ring RNA synthesis at the initiation level“ durch DRB. Vielmehr sieht es so aus, DRB wirkt bei der transkriptionellen Elongation inhibierend. Durch in situ‐Hybridisierung von Sequenzen klonierter BR2‐DNA wurde in Speicheldrüsenchromosom IV das BR2‐Gen in Region 3B9/10 direkt identifiziert. [Sass, H. and Pederson, T. (1984), J. Mol. Biol. 180: 911‐926]. Transcription‐dependent localization of U1 and U2 small nuclear ribonucleoproteins at major sites of gene activity in polytene chromosomes. Immunolokalisation von Sm‐, U1‐ und U2snRNP‐spezifischen Antigenen in Speicheldrüsenchromosomen von C. tentans hat zur Entdeckung der beim Spleißen von prä‐mRNA beteiligten U1/U2snRNPs in Balbianiringen BR2, BR1, BR3 sowie anderen Puffs und aufgelockerten Banden geführt. Die überraschenden BR‐Daten zeigen erstmals: (i) Der Spleiß‐Apparat ist in Genloci mit intensiver RNA‐Synthese schon vorhanden. (ii) Immunfluoreszenz reflektiert den Exon‐Intron‐Bau dieser BR‐Gene. (iii) Transkription und spleißosomales Ausschneiden von Introns sind koordiniert. [Sass, H. (1989), Nucleic Acids Research 17: 10508]. Hsp82‐neo transposition vectors to study insertional mutagenesis in Drosophila melanogaster and tissue culture cells; [Sass, H. (1990), Gene 89: 179‐186]. P‐transposable vectors expressing a constitutive and thermoinducible hsp82‐neo fusion gene for Drosophila germline transformation and tissue‐culture transfection. Beschrieben sind Design, Konstruktion und Expression der Genfusion hsp82‐neo als ein in vivo selektierbares Reporter‐/Markergen, die Transposons P{hsp82‐neo/Adh} sowie P{hsp82‐neo} und Transformations‐Vektoren pHS22, pHS24, pHS85, pHS103 und pHS104. Sie stellen das von der Fliege gebildete Enzym bakteriellen Ursprungs, Neomycin‐Phosphotransferase II, für die G418‐Selektion bereit, um die Position, Struktur, Expression und Funktion von Genen mittels hsp82‐neo‐Mutagenese zu erforschen. [Sass, H. and Meselson, M. (1991), Proc. Natl. Acad. Sci. USA 88: 6795‐6799]. Dosage compensation of the Drosophila pseudoobscura Hsp82 gene and the D. melanogaster Adh gene at ectopic sites in D. melanogaster. Quantitative Unterschiede in der Dosiskompensation des X‐chromosomalen hsp82‐Gens von D. pseudoobscura und autosomalen Adh‐Gens von D. melanogaster wurden als Erhöhung der RNAMenge in D. melanogaster gemessen. Beide Transgene sind dosiskompensiert, sprang P{hsp82‐ neo/Adh} in euchromatische Regionen des D. melanogaster X‐Chromosoms. Beide Transgene sind nicht dosiskompensiert, insertierte P{hsp82‐neo/Adh} ins β‐Heterochromatin in Region 20 an der Basis des X. Keine der zehn autosomalen Insertionen ist dosiskompensiert. Die Ergebnisse lassen vermuten, dass X‐chromosomale regulatorische Sequenzen, die für die Verstärkung der Genaktivität um Faktor 2 in Männchen verantwortlich sind, gehäuft im X vorkommen, jedoch im β‐ Heterochromatin und den Autosomen fehlen. Das Kompensationsverhalten der transponierten Gene wird durch das neue chromosomale Milieu des Insertionsortes bestimmt.

Immunohistochemical localization of RANK, RANKL and OPG in healthy and arthritic canine elbow joints

OBJECTIVE: To determine if the receptor activator of nuclear factor-kappaB-receptor activator of nuclear factor-kappaB ligand-osteoprotegerin (RANK-RANKL-OPG) system is active in bone remodeling in dogs and, if so, whether differences in expression of these mediators occur in healthy and arthritic joints. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease. METHODS: Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints. RESULTS: All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few. CONCLUSIONS: The RANK-RANKL-OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption. CLINICAL RELEVANCE: Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation.

RNA-dependent genome processing during nuclear differentiation: the model systems of stichotrichous ciliates

We introduce ciliated protozoa, and more specifically the stichotrichous ciliates Oxytricha and Stylonychia, as biological model systems for the analysis of programmed DNA-reorganization processes during nuclear differentiation. These include DNA excision, DNA elimination, reordering of gene segments and specific gene amplification. We show that small nuclear RNAs specify DNA sequences to be excised or retained, but also discuss the need for a RNA template molecule derived from the parental nucleus for these processes. This RNA template guides reordering of gene segments to become functional genes and determines gene copy number in the differentiated nucleus. Since the template is derived from the parental macronucleus, gene reordering and DNA amplification are inherited in a non-Mendelian epigenetic manner.

Microscopic analysis of chromatin localization and dynamics in C. elegans

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.