A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina

Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K+ currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells.

Eficacia en los productos cosméticos: un enfoque celular

La regulación europea que define el desarrollo y comercialización de los productos cosméticos hace un énfasis especial en la necesidad de demostrar la efectividad de los productos cosméticos, en un entorno en el que, debido a la prohibición de uso de modelos experimentales animales, sólo es posible el estudio en modelos alternativos (in silico, in vitro, en cultivo, etc.) o en voluntarios sanos. Dentro de los métodos alternativos, la utilización de los modelos celulares adquiere cada vez más importancia por los grandes avances que se producen en la comprensión de los complejos procesos moleculares que regulan y controlan el devenir de la célula, individualmente y dentro de los tejidos.

Caracterização macroscópica das gônadas durante a reprodução induzida em cativeiro do surubim-do-paraíba

O objetivo deste trabalho foi descrever a morfologia das gônadas do surubim-do-paraíba (F1) durante a reprodução induzida em cativeiro. Trinta fêmeas foram induzidas, tendo-se utilizado extrato hipofisário de carpa e gonadotrofina coriônica humana. Vinte e oito fêmeas responderam à indução, tendo liberado 238,80 g de ovócitos (taxa de fertilização: 24,33±8,35%). Após a extrusão dos gametas, os animais foram eutanasiados para análise morfológica das gônadas. Os testículos não apresentaram alterações morfológicas após a indução. Entre os ovários, 63,31% apresentaram má formação (desenvolvimento ímpar, não uniforme ou "afunilamento"), e o número de ovócitos liberados foi abaixo do obtido em reprodutores selvagens desta espécie.

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression ofSSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.

New insights into cellular prion protein (PrPc) functions: the 'ying and yang' of a relevant protein.

The conversion of cellular prion protein (PrPc), a GPI-anchored protein, into a protease-K-resistant and infective form (generally termed PrPsc) is mainly responsible for Transmissible Spongiform Encephalopathies (TSEs), characterized by neuronal degeneration and progressive loss of basic brain functions. Although PrPc is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. Recent studies have confirmed its participation in basic physiological processes such as cell proliferation and the regulation of cellular homeostasis. Other studies indicate that PrPc interacts with several molecules to activate signaling cascades with a high number of cellular effects. To determine PrPc functions, transgenic mouse models have been generated in the last decade. In particular, mice lacking specific domains of the PrPc protein have revealed the contribution of these domains to neurodegenerative processes. A dual role of PrPc has been shown, since most authors report protective roles for this protein while others describe pro-apoptotic functions. In this review, we summarize new findings on PrPc functions, especially those related to neural degeneration and cell signaling.

Nanocharacterization of Soft Biological Samples in Shear Mode with Quartz Tuning Fork Probes

Quartz tuning forks are extremely good resonators and their use is growing in scanning probe microscopy. Nevertheless, only a few studies on soft biological samples have been reported using these probes. In this work, we present the methodology to develop and use these nanosensors to properly work with biological samples. The working principles, fabrication and experimental setup are presented. The results in the nanocharacterization of different samples in different ambients are presented by using different working modes: amplitude modulation with and without the use of a Phase-Locked Loop (PLL) and frequency modulation. Pseudomonas aeruginosa bacteria are imaged in nitrogen using amplitude modulation. Microcontact printed antibodies are imaged in buffer using amplitude modulation with a PLL. Finally, metastatic cells are imaged in air using frequency modulation.

Eficacia en los productos cosméticos: un enfoque celular

La regulación europea que define el desarrollo y comercialización de los productos cosméticos hace un énfasis especial en la necesidad de demostrar la efectividad de los productos cosméticos, en un entorno en el que, debido a la prohibición de uso de modelos experimentales animales, sólo es posible el estudio en modelos alternativos (in silico, in vitro, en cultivo, etc.) o en voluntarios sanos. Dentro de los métodos alternativos, la utilización de los modelos celulares adquiere cada vez más importancia por los grandes avances que se producen en la comprensión de los complejos procesos moleculares que regulan y controlan el devenir de la célula, individualmente y dentro de los tejidos.

Pseudomixoma peritoneal: aspectos tomográficos e na ressonância magnética - relato de três casos

O pseudomixoma peritoneal é um tumor incomum, de curso indolente, que se caracteriza pela presença de ascite mucinosa ou implantes na cavidade peritoneal. Origina-se geralmente de lesões no apêndice ou no ovário. O diagnóstico pode ser feito por meio da citologia de aspiração por agulha fina, ultra-sonografia, tomografia computadorizada ou ressonância magnética. Os autores relatam três casos de pseudomixoma peritoneal cujo sítio primário era o ovário, e que foram submetidos a tomografia computadorizada e a ressonância magnética do abdome. Este trabalho enfatiza a importância destes métodos em função de sua capacidade de resolução espacial, imagens multiplanares e diferentes seqüências (na ressonância magnética), permitindo melhor avaliação das lesões. Os exames tomográficos demonstraram massas lobuladas, hipodensas, com limites bem definidos, determinando "lobulações" nas margens hepática e esplênica por compressão extrínseca secundária a implantes peritoneais, sem invasão dos órgãos. A ressonância magnética revelou lesões expansivas com baixo sinal nas imagens ponderadas em T1 e alto sinal em T2, de localização peritoneal, junto às margens do fígado e baço.

A Feedfordward Adaptive Controller to Reduce the Imaging Time of Large-Sized Biological Samples with a SPM-Based Multiprobe Station

The time required to image large samples is an important limiting factor in SPM-based systems. In multiprobe setups, especially when working with biological samples, this drawback can make impossible to conduct certain experiments. In this work, we present a feedfordward controller based on bang-bang and adaptive controls. The controls are based in the difference between the maximum speeds that can be used for imaging depending on the flatness of the sample zone. Topographic images of Escherichia coli bacteria samples were acquired using the implemented controllers. Results show that to go faster in the flat zones, rather than using a constant scanning speed for the whole image, speeds up the imaging process of large samples by up to a 4x factor.

Tumor de Wilms extra-renal: relato de caso

Um caso de tumor de Wilms extra-renal de localização retroperitoneal em uma paciente do sexo feminino de dois anos de idade é apresentado, associado a revisão de literatura. Foram realizados exames de radiografia simples do abdome, urografia excretora, ultra-sonografia e tomografia computadorizada sem e com contraste, que evidenciaram a presença de massa retroperitoneal adjacente ao rim direito. A paciente foi submetida a intervenção cirúrgica, com ressecção de toda a massa, sendo o diagnóstico de tumor de Wilms confirmado com exame anatomopatológico. O tumor de Wilms extra-renal é uma entidade extremamente rara e maligna, descrito na literatura principalmente sob a forma de relato de caso. Pode ocorrer no retroperitônio, útero e ovários, canal inguinal, testículos, pele, e até mesmo no tórax. O mecanismo exato que poderia explicar a ocorrência deste tumor em tecido extra-renal não foi bem estabelecido ainda. O diagnóstico é feito através do estudo anatomopatológico da lesão, geralmente após intervenção cirúrgica.

Aspectos ultra-sonográficos das alterações não-neoplásicas do testículo

Processos patológicos dos testículos são muito comuns, incluindo-se lesões tumorais e não-tumorais neste contexto. A ultra-sonografia com transdutor de alta freqüência tornou-se a modalidade de imagem de escolha para a avaliação desses órgãos. Este método ajuda a melhor caracterizar lesões intratesticulares e em muitas situações sugere um diagnóstico mais específico, principalmente nos casos em que há manifestações clínicas similares, tais como dor, inchaço e aumento volumétrico locais. O mapeamento com Doppler colorido é importantíssimo para demonstrar padrões anormais de perfusão testicular e auxilia no diagnóstico de condições clínicas agudas. Neste ensaio iconográfico os autores sumarizam os mais comuns achados clínicos, patológicos e as principais características diagnósticas de lesões testiculares, tais como microlitíase, cisto simples, espermatocele, varicocele, ectasia tubular da rete testis, orquite, hematomas e condições mais raras. A familiaridade com as características ecográficas e clínicas destas alterações é essencial para o estabelecimento do diagnóstico correto e início da terapêutica mais eficaz, quando necessária.

Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells

Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.

Competition in notch signaling with cis enriches cell fate decisions

Notch signaling is involved in cell fate choices during the embryonic development of Metazoa. Commonly, Notch signaling arises from the binding of the Notch receptor to its ligands in adjacent cells driving cell-to-cell communication. Yet, cell-autonomous control of Notch signaling through both ligand-dependent and ligand-independent mechanisms is known to occur as well. Examples include Notch signaling arising in the absence of ligand binding, and cis-inhibition of Notch signaling by titration of the Notch receptor upon binding to its ligands within a single cell. Increasing experimental evidences support that the binding of the Notch receptor with its ligands within a cell (cis-interactions) can also trigger a cell-autonomous Notch signal (cis-signaling), whose potential effects on cell fate decisions and patterning remain poorly understood. To address this question, herein we mathematically and computationally investigate the cell states arising from the combination of cis-signaling with additional Notch signaling sources, which are either cell-autonomous or involve cell-to-cell communication. Our study shows that cis-signaling can switch from driving cis-activation to effectively perform cis-inhibition and identifies under which conditions this switch occurs. This switch relies on the competition between Notch signaling sources, which share the same receptor but differ in their signaling efficiency. We propose that the role of cis-interactions and their signaling on fine-grained patterning and cell fate decisions is dependent on whether they drive cis-inhibition or cis-activation, which could be controlled during development. Specifically, cis-inhibition and not cis-activation facilitates patterning and enriches it by modulating the ratio of cells in the high-ligand expression state, by enabling additional periodic patterns like stripes and by allowing localized patterning highly sensitive to the precursor state and cell-autonomous bistability. Our study exemplifies the complexity of regulations when multiple signalng sources share the same receptor and provides the tools for their characterization.

Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration (in press)

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.

Vaporização a laser do cervix para tratamento da neoplasia intraepitelial cervical

O câncer cérvico-uterino é muito comum em vários países da América Latina. As estatísticas de mortalidade e as taxas de incidência demonstram a sua real importância. O cânver cérvico-uterino freqüentemente é uma doença progressiva iniciada com mudanças intra-epiteliais, que podem se transformar em um processo invasivo, sendo o nosso objetivo tratar precocemente estas lesões quando ainda é possível a cura de 100%. Em nosso estudo prospectivo foram selecionadas 21 pacientes com neoplasia cervical intra-epitelial reatreadas pela citplogia e diagnosticadas pela histopatologia após biópsia dirigida pela colposcopia. O método terapêutico empregado foi a vaporização a laser com o CO2. Tiveram como pré-requisito os seguintes critérios: informação segura pela colposcopia da zona de transformação e afastar a presença de câncer invasivo; a neoplasia epitelial cervical deve ocupar a ectocervix sem nenhuma extensão para o canal cervical e correlação positiva entre a citologia, colposcopia e histologia. O uso de laser CO2 com microscópio permitiu precisão na aplicação e com vantagens de ser um procedimento ambulatorial diminuindo estresse cirúrgico das pacientes. Foi realizado sem anestesia e com duração média de 15 minutos. A cicatrização completou-se em torno de três semanas e com cuidados operatórios mínimos. Somente dois casos tiveram sangramento vaginal discreto no quinto e décimo dia de pós-operatório, resolvido com tamponamento vaginal por 24 horas. A colposcopia, cirurgia e o seguimento foram feitos pelo autor, tendo uma paciente sido submetida a uma segunda vaporização no quinto mês de controle. Somente uma paciente teve recidiva no 26° mês de seguimento e complementará o tratamento. As vinte outras restantes estão em controle sem recidiva de doença. Em vista dos resultados obtivemos um percentual de cura de 95%, que coincide com a literatura. O uso de laser CO2 no tratamento das neoplasias cervicais intra-epiteliais (NIC) ou virais tem sido estimulado como uma alternativa digna de ser seguida pelas seguintes razões: cirurgia de não contato, tratamento rápido e indolor, diminuição das custas da internação; complicações mínimas e sem efeito subseqüente sobre a fertilidade e competência cervical, menor necrose térmica, e possibilidade de novo tratamento ambulatorial. Por estas razões e pelo alto percentual de cura podemos concluir que a cirurgia proposta foi vantajosa para o tratamento da neoplasia cervical intra-epitelial, quando comparada com outros métodos de tratamento.