Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines

Aim: It has been demonstrated that the endogenous matrix metalloproteinases (MMPs) inhibitor reversion inducing cysteine rich protein with Kazal motifs (RECK) is a reliable prognostic marker for detecting several types of tumors. However, the RECK expressions in most of the normal and neoplastic tissues were extremely low, and to measure its expression is quite complicated. The purpose of the present study is to establish an easy method to quantify murine RECK mRNA expression for use in future experimental studies. Subsequently, in order to verify the reliability of the established quantification technique, we examined the change in RECK expression and gelatinase secretion in tumor cells when stimulated by the extracellular matrix. Methods: Several murine tumor cells were used in the present study. The real-time polymerase chain reaction (PCR) method and measurement conditions for murine RECK mRNA were studied using these tumor cells. Gelatinase activities were also examined by gelatin zymography. Results: Murine RECK mRNA expression was accurately quantified using real-time PCR. Among the tumor cells used in the study, osteosarcoma cells showed significantly higher RECK mRNA expression than the others. The RECK expression in the osteosarcoma cells was down-regulated by contact with matrigel-coated culture flasks due to increased secretion of gelatinases. Conclusion: The real-time PCR method employed in our study is useful to quantify RECK expression.Показано, что эндогенный ингибитор матриксных протеиназ (MMП) RECK может служить надежным прогностическим маркером для некоторых типов опухолей, однако его экспрессия в большинстве нормальных и неопластических тканей крайне низкая, поэтому возникают сложности, связанные с детекцией таковой. Цель работы — разработка количественного метода определения экспрессии мРНК для использования в экспериментальных исследованиях. Для дальнейшего подтверждения надежности разработанного метода исследованы изменения экспрессии RECK и секреции желатиназ в опухолевых клетках при стимуляции внеклеточным матриксом. Методы: в работе использовали несколько линий опухолевых клеток мыши, в которых экспрессию мРНК RECK анализировали методом ПЦР в режиме реального времени, активность желатиназ — методом зимографии. Результаты: экспрессию мРНК RECK количественно оценили методом ПЦР в режиме реального времени, причем среди исследованных клеточных линий наиболее высокий уровень экспрессии RECK выявили в клетках остеосаркомы. Экспрессия RECK в клетках остеосаркомы подавлялась при контакте с культуральным пластиком, обработанным матригелем, вследствие повышения секреции желатиназ. Выводы: для количественной оценки экспрессии мРНК RECK может быть использован метод ПЦР в режиме реального времени

Amamentação em Prematuros Investigação do Significado Materno

O bebê que nasce prematuro necessita de cuidados diferenciados. A permanência na Unidade de Terapia Intensiva Neonatal é circundada de acontecimentos novos e cuidados especiais, entre eles, a amamentação. A mãe de um bebê nascido antes do tempo previsto tem reduzido o período de preparação para recebê-lo. O objetivo foi investigar e analisar o signifi cado de amamentar um recémnascido pré-termo, atribuído por uma mãe, que teve seu bebê atendido em Unidade de Terapia Intensiva Neonatal em um município do Paraná. O estudo foi descritivo, de abordagem qualitativa. Foi realizado a partir de uma entrevista aberta em grupo focal. A fala de uma das participantes foi recortada, transcrita e analisada a partir de análise do discurso, modalidade temática. A fala da mãe revelou sentimentos, ora favoráveis referentes à importância da amamentação, ora desfavoráveis referentes à obrigação em aleitar e, algumas vezes, ambíguos, em relação ao signifi cado da amamentação em ambiente de Unidade de Terapia Intensiva Neonatal. É imprescindível uma atenção especial às mães nesta condição para que haja sucesso na amamentação durante a internação e no momento de alta clínica do bebê

Aplicação de um Instrumento de Avaliação da Prontidão do Prematuro para Início da Alimentação Oral: Estudo Descritivo

Descrever o comportamento do prematuro mediante aplicacao de um instrumento de avaliacao da prontidao do bebe para iniciar sua alimentacao por via oral, e o objetivo desse trabalho, desenvolvido por meio de estudo descritivo exploratorio. O instrumento de avaliacao foi aplicado em 60 bebes prematuros. Pode-se verifi car que a maioria dos bebes estudados possuia idade gestacional corrigidamaior ou igual a 34 semanas, apresentou estado comportamental sono leve, adequacao quanto a postura e tonus global, adequacao da postura de labios, presenca dos refl exos de mordida e vomito, movimento adequado de lingua, presenca do canolamento de lingua e adequacao dos movimentos de mandibula. Todos os bebes avaliados apresentaram postura de lingua plana. Ao analisar a presenca do refl exo de procura, a maioria dos bebes nao o apresentou. Quanto ao refl exo de succao, todos os bebes apresentaram este refl exo, sendo metade de forma debil. A forca de succao mostrou-se adequada em 51,7% dos casos e fraca em 45,6%. O ritmo de succao por pausa mais comum foi o numero de succoes menor do que 5, seguido do grupo entre 5 a 8 succoes. Percebeu-se, neste estudo, que a maioria dos bebes não manteve o ritmo de succao por pausa e a maior parte dos bebes manteve parcialmente o estado comportamental alerta apos avaliacao da succao nao-nutritiva

P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress

DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.Peer reviewe

Botulinum Neurotoxins and Botulism: A Novel Therapeutic Approach

Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15–20 kDa single domain antibody (VHH) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/VHH phage display library. The VHH has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the VHH and the toxin but also an insertion of the VHH CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the VHH to a cell penetrating peptide (CPP), the CPP-VHH fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme