Karyotypic diversity in four species of the genus Gymnotus Linnaeus, 1758 (Teleostei, Gymnotiformes, Gymnotidae): physical mapping of ribosomal genes and telomeric sequences

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mapeamento físico de genes ribosomais 18S e 5S nos cromossomos de espécies simpáticas do gênero Gymnotus (Teleostei, Gymnotiformes, Gymnotidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A terbenzimidazole that preferentially binds and conformationally alters structurally distinct DNA duplex domains: A potential mechanism for topoisomerase I poisoning

The terbenzimidazoles are a class of synthetic ligands that poison the human topoisomerase I (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by binding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. To test this hypothesis, we characterize and compare the binding properties of a 5-phenylterbenzimidazole derivative (5PTB) to the d(GA4T4C)2 and d(GT4A4C)2 duplexes. The d(GA4T4C)2 duplex contains an uninterrupted 8-bp A⋅T domain, which, on the basis of x-ray crystallographic data, should induce a highly hydrated “A-tract” conformation. This duplex also exhibits anomalously slow migration in a polyacrylamide gel, a feature characteristic of a noncanonical global conformational state frequently described as “bent.” By contrast, the d(GT4A4C)2 duplex contains two 4-bp A⋅T tracts separated by a TpA dinucleotide step, which should induce a less hydrated “B-like” conformation. This duplex also migrates normally in a polyacrylamide gel, a feature further characteristic of a global, canonical B-form duplex. Our data reveal that, at 20°C, 5PTB exhibits an ≈2.3 kcal/mol greater affinity for the d(GA4T4C)2 duplex than for the d(GT4A4C)2 duplex. Significantly, we find this sequence/conformational binding specificity of 5PTB to be entropic in origin, an observation consistent with a greater degree of drug binding-induced dehydration of the more solvated d(GA4T4C)2 duplex. By contrast with the differential duplex affinity exhibited by 5PTB, netropsin and 4′,6-diamidino-2-phenylindole (DAPI), two AT-specific minor groove binding ligands that are inactive as human TOP1 poisons, bind to both duplexes with similar affinities. The electrophoretic behaviors of the ligand-free and ligand-bound duplexes are consistent with 5PTB-induced bending and/or unwinding of both duplexes, which, for the d(GA4T4C)2 duplex, is synergistic with the endogenous sequence-directed electrophoretic properties of the ligand-free duplex state. By contrast, the binding to either duplex of netropsin or DAPI induces little or no change in the electrophoretic mobilities of the duplexes. Our results demonstrate that the TOP1 poison 5PTB binds differentially to and alters the structures of the two duplexes, in contrast to netropsin and DAPI, which bind with similar affinities to the two duplexes and do not significantly alter their structures. These results are consistent with a mechanism for TOP1 poisoning in which drugs such as 5PTB differentially target conformationally distinct DNA sites and induce structural changes that promote enzyme-mediated DNA cleavage.

Morphological characterization of two new species of Dicontophrya (Ciliophora: Astomatia: Contophryidae) commensal of earthworms (Oligochaeta: Annelida) of ebebda and nkolbikogo (Cameroon)

Article

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. Results. We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4,6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. Conclusions. These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.

Imunohistoquímica no diagnóstico da glomerulonefrite membranosa: estudo comparativo de binómios cromogênio + coloração de contraste

A glomerulonefrite membranosa faz parte das doenças glomerulares que provocam glomerulonefrite crônica, apresentando-se como uma das causas da doença renal terminal. As técnicas de imunofluorescência são o gold standard no estudo imunológico desta patologia em biópsia renal, através da deteção de imunocomplexos (e.g. IgG e C3) e do seu padrão de distribuição granular característico. No entanto, a imunofluorescência não permite uma contextualização histológica e os fluorocromos utilizados possuem um reduzido tempo de atividade, ao contrário das técnicas imunoenzimáticas que utilizam cromogénios coloridos precipitados que permitem a obtenção de uma marcação permanente e a sua contextualização histológica por via da utilização de eficientes colorações de contraste. Com a finalidade de contribuir para a qualidade do diagnóstico da glomerulonefrite membranosa, em biópsias renais, procurou-se, com esta pesquisa, identificar uma técnica imunoenzimática, através da conjugação entre diferentes cromogênios e colorações de contraste, que permita a deteção de depósitos de IgG e C3, com padrão granular. Foram constituídos diferentes binômios cromogênio + coloração, com os cromogênios 3,3›- Diaminobenzidine Tetrahydrochloride e 3-Amino-9-ethylcarbazole e as colorações Periodic Acid Schiff, Periodic Acid Methenamine Silver e Hematoxilina. Foram utilizadas 72 secções de tecido provenientes de seis de casos de biópsias renais com diagnóstico de glomerulonefrite membranosa, fixados em formalina a 10% e incluídos em parafina. A recolha de dados foi realizada por observação microscópica com preenchimento de uma grelha de classificação dos parâmetros: preservação da morfologia, intensidade da marcação específica, quantidade relativa de estruturas marcadas, marcação inespecífica/fundo, contraste e padrão da marcação, que permitiu a classificação dos binómios estudados num score quantitativo de 0-100 pontos. O binômio que apresentou melhores resultados foi 3-Amino-9-ethylcarbazole + Hematoxilina (score 71,81) e o binômio 3,3›- Diaminobenzidine Tetrahydrochloride+Periodic Acid Methenamine Silver (score 7,81), apresentou os piores resultados. O resultado do teste Kruskal-Wallis indica-nos a presença de diferenças estatísticas entre os binómios em estudo (p=0,000). A Hematoxilina pode ser considerada a coloração mais eficaz, pois cumpriu a sua função de auxiliar e facilitar a observação do tipo de padrão com os dois cromogênios utilizados. O cromogênio 3-Amino-9-ethylcarbazole apresentou resultados semelhantes aos produzidos pelo 3,3›-Diaminobenzidine Tetrahydrochloride, no entanto, permitiu identificar em todos os casos o padrão granular de imunomarcação, ao contrário do que aconteceu com este último.

Characterization of invasive fish species in a river transposition region: evolutionary chromosome studies in the genus Hoplias (Characiformes, Erythrinidae)

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

Molecular cytogenetic study of heterochromatin in Hisonotus leucofrenatus (Teleostei, Loricariidae, Hypoptopomatinae)

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

Cytogenetics of the Brazilian Bolitoglossa paraensis (Unterstein, 1930) salamanders (Caudata, Plethodontidae)

Plethodontid salamanders of genus Bolitoglossa constitute the largest and most diverse group of salamanders, including around 20% of living caudate species. Recent studies have indicated the occurrence of five recognized species in the Brazilian Amazon Rainforest. We present here the first cytogenetic data of a Brazilian salamander, which may prove to be a useful by contribution to the cytotaxonomy of the genus. Specimens were collected near the “type” locality (Utinga, Belém, PA, Brazil). Chromosomal preparations from duodenal epithelial cells and testes were subjected to Giemsa staining, C-banding and DAPI/CMA₃ fluorochrome staining. All specimens showed a karyotype with 13 bi-armed chromosome pairs (2n = 26). Nucleolar Organizer Regions, evidenced by CMA₃, were located distally on the long arm of pair 7 (7q). DAPI₊ heterochromatin was predominantly centromeric, with some small pericentromeric bands. Although the C-banding patterns of other Bolitoglossa species are so far unknown, cytogenetic studies conducted in other Plethodontid salamanders have demonstrated that pericentromeric heterochromatin is a useful cytological marker for identifying interspecific homeologies. Species diversification is usually accompanied by chromosomal changes. Therefore, the cytogenetic characterization of Bolitoglossa populations from the middle and western Brazilian Amazon Basin could identify differences which may lead to the identification of new species.

Análise cariotípica de duas espécies de cigarrinhas do gênero Mahanarva (Hemiptera, Cercopidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise cariotípica de duas espécies de cigarrinhas do gênero Mahanarva (Hemiptera, Cercopidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Isolierung und Identifizierung von wachstumsfördernden Substanzen für das weinrelevante Bakterium Oenococcus oeni aus Fruchtsäften und Blattextrakten

Neben Tomatensaft wurde eine Vielzahl von Säften und Blattextrakten als Medienzusätze auf Wachstumsförderung bei 30 verschiedenen Oenococcus oeni-Stämmen getestet. Es zeigte sich eine breite Wachstumsförderung bei allen Zusätzen mit Ausnahme von Zitronensaft, sodass die Wachstumsfaktoren keine tomatenspezifischen Inhaltsstoffe sein können und eher ubiquitär in der Pflanzenwelt vorkommen. Das Ausmaß der Wachstumsförderung war stammabhängig sehr unterschiedlich und Tomatensaft stellte keineswegs für alle Stämme den optimalen Medienzusatz dar. Durch Schälen der Früchte war eine für die Analytik hilfreiche Abtrennung schalenspezifischer Inhaltsstoffe möglich, wobei auch die Schalenextrakte großes Potential für die Suche nach Wachstumsfaktoren offenbarten und die Wichtigkeit einer Auftrennung der Frucht in die verschiedenen Fruchtbereiche betonte. Aus Tomatensaft konnte analytisch der anorganische Wachstumsfaktor Mangan identifiziert werden. Die größten Zelldichten der Oenokokken-Stämme wurden hierbei bei 67 µM und 34 mM Manganzusatz erreicht. Bei 13 von 20 getesteten Oenokokkenstämmen konnte bei Zusatz von 34 mM Mangan der Tomatensaft ersetzt werden, bei 4 Stämmen (z. B. Stamm B2) fehlten jedoch noch weitere Wachstumsfaktoren und bei 3 Stämmen (z. B. Stamm B120) kam es zu einem verfrühten Absterben. Da weitere Mineralstoffe sowie veraschte Säfte und Blattextrakte keinen positiven Einfluß auf die Oenokokken-Zelldichte hatten, wurde mittels semipräparativer HPLC nach zusätzlichen organischen Wachstumsfaktoren für den Stamm B2 gesucht. Hierzu wurde der nachfolgende Wachstums-Assay miniaturisiert und erfolgreich auf Microtiterplatten etabliert. Es gelang die Isolierung und Identifizierung eines wachstumsfördernden Trisaccharides aus Mangoschalen-Extrakt, das aus den Zuckern Glucose, Rhamnose und Arabinose bestand. Von den monomeren Zuckern erhöhte lediglich die Arabinose die Zelldichte, das Optimum lag bei 1,5 g/l. Auch aus Zitronenmesokarp-Extrakt war die Isolierung eines wachstumsfördernden arabinosehaltigen Disaccharides möglich, die Menge reichte jedoch noch nicht für eine genaue Identifizierung aus. Desweiteren erwies sich 1,5 g/l Cystein als wachstumsstimulierend. Ein Zusatz aller gefundenen Wachstumsfaktoren (34 mM Mangan, 1,5 g/l Arabinose und 1,5 g/l Cystein) ersetzte den Tomatensaft bei weiteren Oenokokken-Stämmen (z.B. Stamm B120) komplett, wobei bei allen Stämmen sogar eine schnellere Anzucht erfolgte. Neben dem Tomatensaft war auch der Zusatz von Hefeextrakt zum Grundmedium nicht mehr nötig, sodass ein neues vereinfachtes Medium für die Anzucht von Oenokokken mit komplexen Nährstoffansprüchen vorgeschlagen werden konnte. Lediglich beim Stamm B2 zeigte sich noch ein OD-Unterschied von 0,2 in der stationären Phase, der nach Adsorptionsversuchen an Polyvinylpolypyrrolidon auf noch unidentifizierte Polyphenole im Tomatensaft zurückzuführen ist. Aus grünem Tee erwies sich das Polyphenol Epigallocatechingallat (EGCG) konzentrationsabhängig sowohl als Hemmstoff (>550 mg/l EGCG) als auch Wachstumsfaktor (400-500 mg/l EGCG) für den Oenokokken-Stamm B2. Der hemmende als auch der fördernde Einfluss auf das Wachstum wurde mittels Sytox/DAPI-Färbung bestätigt. Der sogenannte „Tomatensaft-Faktor“ ist also nicht eine spezielle Substanz, sondern das synergistische Zusammenwirken mehrerer einfacher Substanzen wie Mineralstoffe, Aminosäuren, Kohlenhydrate und Polyphenole. Auch sind die Oenokokken-Stämme bezüglich ihres Nährstoffbedarfes sehr unterschiedlich, sodass für jeden Stamm einzeln das optimale Substratspektrum ermittelt werden muss.

Biogeochemical measurements associated to deep-sea wood falls

Large organic food falls to the deep sea - such as whale carcasses and wood logs - support the development of reduced, sulfidic niches in an otherwise oxygenated, oligotrophic deep-sea environment. These transient hot spot ecosystems may serve the dispersal of highly adapted chemosynthetic organisms such as thiotrophic bivalves and siboglinid worms. Here we investigated the biogeochemical and microbiological processes leading to the development of sulfidic niches. Wood colonization experiments were carried out for the duration of one year in the vicinity of a cold seep area in the Nile deep-sea fan (Eastern Mediterranean) at depths of 1690 m. Wood logs were deployed in 2006 during the BIONIL cruise (RV Meteor M70/2 with ROV Quest, Marum, Germany) and sampled in 2007 during the Medeco-2 cruise (RV Pourquoi Pas? with ROV Victor 6000, Ifremer, France). Wood-boring bivalves played a key role in the initial degradation of the wood, the dispersal of wood chips and fecal matter around the wood log, and the provision of colonization surfaces to other organisms. Total oxygen uptake measured with a ROV-operated benthic chamber module was higher at the wood (0.5 m away) in contrast to 10 m away at a reference site (25 mmol m-2 d-1 and 1 mmol m-2 d-1, respectively), indicating an increased activity of sedimentary communities around the wood falls. Bacterial cell numbers associated with wood increased substantially from freshly submerged wood to the wood chip/fecal matter layer next to the wood experiments, as determined with Acridine Orange Direct Counts (AODC) and DAPI-stained counts. Microsensor measurements of sulfide, oxygen and pH were conducted ex situ. Sulfide fluxes were higher at the wood experiments when compared to reference measurements (19 and 32 mmol m-2 d-1 vs. 0 and 16 mmol -2 d-1, respectively). Sulfate reduction (SR) rates at the wood experiments were determined in ex situ incubations (1.3 and 2.0 mmol m-2 d-1) and fell into the lower range of SR rates previously observed from other chemosynthetic habitats at cold seeps. There was no influence of wood deposition on phosphate, silicate and nitrate concentrations, but ammonium concentrations were elevated at the wood chip-sediment boundary layer. Concentrations of dissolved organic carbon were much higher at the wood experiments (wood chip-sediment boundary layer) in comparison to measurements at the reference sites, which may indicate that cellulose degradation was highest under anoxic conditions and hence enabled by anaerobic benthic bacteria, e.g. fermenters and sulfate reducers. Our observations demonstrate that, after one year, the presence of wood at the seafloor had led to the creation of sulfidic niches, comparable to what has been observed at whale falls, albeit at lower rates.

Desenvolvimento floral e do óvulo e aspectos da reprodução em Aechmea sp. e Vriesea sp. (Bromeliaceae)

A utilização de Bromélias tem sido crescente no mercado de plantas onamentais, por outro lado, muitas espécies encontram-se ameaçadas, grande parte pelos impactos humanos no ambiente. Aechmea correia-araujoi E. Pereira & Moutinho, Aechmea gamossepala Wittm, Vriesea ensiformis (Vell.) Beer e Vriesea saundersii (Carrière) E. Morren ex Mez, espécies nativas da Mata Atlântica brasileira, têm sido alvo de extrativismo. Informações básicas sobre a espécie são essenciais para subsidiar a condução de programas de conservação e melhoramento genético, que aliados a ferramentas biotecnológicas permitem a incorporação de estratégias inovadoras aos métodos de melhoramento. Neste sentido, o objetivo do presente trabalho foi descrever essas espécies, quanto à micromorfologia floral, aspectos reprodutivos envolvidos no processo de polinização, desenvolvimento floral e deesenvolvimento gametofítico, como mecanismo de preservação e produção comercial. A caracterização morfológica e anatômica das flores das espécies de Aechmea e Vriesea contribuiu para a compreensão do processo reprodutivo. As espécies apresentam grãos de pólen com alta capacidade reprodutiva, viabilidade polínica superior a 93%, germinação in vitro maior que 80% e o estigma apresenta-se receptivo da antese ao final do dia. A ontogênese floral de A. correia-araujoi é centrípeta, os primórdios desenvolvem-se na ordem, sépala, pétala, androceu e gineceu. O apêndice petalar é formado na fase final do desenvolvimento. O primórdio de óvulo tem origem placentária e caráter trizonal, o óvulo é anátropo, bitegumentado e crassinucelado. O meristema floral de A. gamosepala se desenvolve de forma centrípeta, de forma unidirecional reversa. O estigma diferencia-se na fase inicial do desenvolvimento e os apêndices petalares, na fase final. O óvulo é anátropo, crassinucelado, bitegumentado, tétrade linear, megásporo calazal funcional, desenvolvimento tipo monospórico e Polygonum. As anteras são bitecas, tetraesporangiadas, com tapete secretor. Botões florais de 8,7 - 13,0 mm são indicados no estudo de embriogênese a partir de micrósporo. As alterações celulares e o padrão de distribuição de pectinas e AGPs foram caracterizadas por análise citoquímica com azul de toluidina, KI e DAPI e imunocitoquímica por imunofluorescência com os anticorpos para RNA, pectinas esterificadas (JIM7), não esterificadas (JIM5) e AGPs (LM2, LM6, MAC207, JIM13, JIM14) e analisadas por microscopia de fluorescência. Foram caracterizados padrões de distribuição espaço-temporal de pectinas e AGP que podem ser utilizados como marcadores de desenvolvimento gametofítico masculino. As observações feitas nesse trabalho fornecem dados sobre aspectos reprodutivos das espécies que podem ser utilizados em programas de melhoramento genético, conservação e desenvolvimento de haploides

Cellular interactions with hydrogels

An efficient means of evaluating potential biomaterials is to use the in vitro fibroblast cell culture model. However, the chemistry which influences cell adhesion on polymer substrates is poorly understood. The work in this thesis aims to rationalise several theories of current opinion and introduce new chemical techniques that may predict cellular behaviour. The keratoprosthesis is a typical example of the need to be able to manipulate cell adhesion of materials since both adhesive and non adhesive sections are needed for proper integration and optical function. Calcein AM/ethidium homodimer-1 and DAPI assays were carried out using 3T3 and EKl.BR cells. Poly(HEMA) was found to be the most cell adhesive hydrogel tested. The reactivity of monomers and the resulting sequence distribution were found to affect surface properties and this may explain the poor levels of cell adhesion seen on NVP/MMA copolymers. Surface free energy is shown to be dependent on the polar and non polar groups present along the backbone chain of the polymers. Dehydrated and hydrated contact angle measurements show the effect of rotation of surface groups around the backbone chain. This effect is most apparent on hydrogels containing methacrylic acid. Dynamic contact angle measurements confirm sequence distribution irregularities and demonstrate the mobility of surface groups. Incorporation of NVI or DEAEMA into the hydrogels does not affect the mobility of the surface groups despite their bulkiness. Foetal calf serum was used for the first time as a test solution in an attempt to mimic a biological environment during surface experiments. A Vroman effect may be present, and may involve different surface proteins for each material tested. This interdisciplinary study combines surface characterisation and biological testing to further the knowledge of the biomaterial/host interface. Surface chemistry techniques appear to be insufficiently sensitive to predict cellular behaviour. The degree of ionisation of hydrogels containing ionic groups depends on the nature of the functional groups as well as the concentration and this is an important parameter to consider when comparing charged materials.