Estudo da adesão, sobrevivência e tubulogênese endotelial em modelo de células de glioma silenciadas para a expressão de Tenascina-C

Recentemente, nosso grupo demonstrou que a matriz extracelular de astrocitomas promove a seleçãode células endoteliais altamente proliferativas, porém com reduzida capacidade tubulogênica, além de determinar a morte de uma segunda sub-população endotelial, por desaderência ou anoikis. Estratégias de simulação dos teores de tenascina-C (TN-C) e fibronectina (FN) nas matrizes de astrocitomas, realizados com ambas as proteínas purificadas na forma de substratos definidos, sugeriram que o balanço TN-C:FN estava relacionado com os fenótipos endoteliais observados. No entanto, este procedimento não permitia abordar a participação de outros componentes da matriz tumoral nativa neste processo. Com objetivo de estudar a modulação do fenótipo angiogênico das células endoteliais por matrizes de astrocitoma, realizamos o silenciamento da expressão de TN-C na linhagem de astrocitoma U-373 MG. O silenciamento foi confirmado por western blotting, PCR em tempo real e ELISA, que permitiram concluir que, no período pós-transfecção (120h) necessário para se obter matrizes tumorais nativas para ensaios funcionais com células endoteliais, as células U-373 MG mantiveram-se silenciadas em índices superiores a 90%. A diminuição de TN-C nas matrizes tumorais resultou em um pequeno (≅18%, em média), porém significativo aumento na taxa de adesão endotelial. HUVECs incubadas com a matriz secretadas por células silenciadas apresentaram uma redução de ≅35% do número de núcleos picnóticos, quando comparadas a HUVECs incubadas com a matriz de células U-373 MG (selvagens ou transfectadas com siRNA controle). O silenciamento da expressão da TN-C na matriz nas células U-373 MG restaurou ainda o defeito tubulogênico das células endoteliais, que passaram a apresentar formação de tubos comparável à obtida quando HUVECs foram incubadas com sua matriz autóloga, rica em FN. Tais resultados apoiam observações anteriores do grupo, que já sugeriam que a maior proporção de FN na matriz autóloga, comparada a matriz do astrocitoma, seria o fator principal para a seleção dos fenótipos angiogênicos observados, demonstrando mais uma vez a importância do balanço FN:TN-C na regulação de processos angiogênicos. Dados anteriores sugeriam ainda que a sub-população endotelial que morre por anoikisapós contato prolongado (24 horas) com matrizes de astrocitomas corresponde a células que já haviam entrado na fase S do ciclo celular, no início da incubação. A fim de nos aprofundarmos sobre a participação do ciclo celular neste processo, a expressão da proteína p27, um inibidor de quinases dependentes de ciclinas (CKI), também foi analisada. HUVECs incubadas com a matriz de astrocitoma apresentaram um aumento de 2 a 3 vezes na expressão de p27, quando comparada com HUVECs provenientes de sua matriz autóloga. No entanto, células endoteliais incubadas com matriz secretada por células U-373 MG silenciadas apresentaram um nível de expressão de p27 comparável ao das HUVECs incubadas com matriz secretada por células selvagens, indicando que a expressão de TN-C não modula, ou não está diretamente correlacionada à expressão da proteína p27. Este resultado sugere que outros componentes da matriz tumoral devam estar envolvidos na modulação do ciclo celular endotelial.

Estudo de polimorfismos nos genes TP53 e p21(WAF1) e do perfil imunohistoquímico das proteínas p53, p21(WAF1), p16(INK4a) e ciclina D1 pela técnica de Tissue Microarray (TMA) e sua importância para o desenvolvimento e/ou severidade das neoplasias cervicais

O câncer de colo do útero é o terceiro tipo de câncer mais frequente em mulheres no mundo, e a infecção persistente pelo papilomavirus humano (HPV) oncogênico é condição necessária, mas não suficiente para seu desenvolvimento. As oncoproteínas virais E6 e E7 interferem direta ou indiretamente na ação de várias proteínas celulares. Entretanto, as variantes proteicas, resultantes de polimorfismos genéticos, podem apresentar comportamento distinto mediante a infecção pelo HPV. O objetivo deste estudo foi avaliar possíveis associações entre polimorfismos nos genes TP53 (p53 PIN3, p53 72C>G) e p21 (p21 31C>A) e o desenvolvimento de neoplasias cervicais, considerando os níveis de expressão das proteínas p53, p21, p16 e ciclina D1, e fatores de risco clássicos para o câncer cervical. Foram selecionadas 466 mulheres residentes no Rio de Janeiro, 281 com diagnóstico histopatológico de neoplasia cervical de baixo (LSIL) e alto grau (HSIL) e câncer (grupo de casos) e 185 sem história atual ou pregressa de alteração citológica do colo uterino (grupo controle). A técnica de PCR-RFLP (reação em cadeia da polimerase - polimorfismo de comprimento de fragmento de restrição), foi empregada na análise dos polimorfismos p53 72C>G e p21 31C>A, usando as enzimas de restrição BstUI e BsmaI, respectivamente. A avaliação do polimorfismo p53 PIN3 (duplicação de 16 pb) foi feita por meio da análise eletroforética direta dos produtos de PCR. A expressão das proteínas p53, p21, p16, ciclina D1 e Ki-67 e a pesquisa de anticorpos anti-HPV 16 e HPV pool foram avaliadas por imunohistoquímica (Tissue Microarray - TMA) em 196 biópsias do grupo de casos. O grupo controle se mostrou em equilíbrio de Hardy-Weinberg em relação aos três polimorfismos avaliados. As distribuições genotípicas e alélicas relativas a p53 PIN3 e p53 72C>G nos grupos controles e de casos não apresentaram diferenças significativas, embora o genótipo p53 72CC tenha aumentado o risco atribuído ao uso de contraceptivos das pacientes apresentarem lesões mais severas (OR=4,33; IC 95%=1,19-15,83). O genótipo p21 31CA(Ser/Arg) conferiu proteção ao desenvolvimento de HSIL ou câncer (OR=0,61, IC 95%=0,39-0,97), e modificou o efeito de fatores de risco associados à severidade das lesões. A interação multiplicativa de alelos mostrou que a combinação p53 PIN3A1, p53 72C(Pro) e p21 31C(Ser), representou risco (OR=1,67, IC95%=1,03-2,72) e a combinação p53 PIN3A1, p53 72C(Pro) e p21 31A(Arg) conferiu efeito protetor (OR=0,26, IC95%=0,08-0,78) para o desenvolvimento de HSIL e câncer cervical. Observou-se correlação positiva da expressão de p16 e p21 e negativa da ciclina D1 com o grau da lesão. A distribuição epitelial de p16, Ki-67, p21 e p53 se mostrou associada à severidade da lesão. Os polimorfismos analisados não apresentaram associação com a expressão dos biomarcadores ou positividade para HPV. Nossos resultados sugerem a importância do polimorfismo p21 31C>A para o desenvolvimento das neoplasias cervicais e ausência de correlação dos polimorfismos p53 PIN3 e p53 72C>G com a carcinogênese cervical, embora alguns genótipos tenham se comportado como modificadores de risco. Nossos resultados de TMA corroboram o potencial de uso de biomarcadores do ciclo celular para diferenciar as lesões precursoras do câncer cervical.

Avaliação da ação antitumoral in vitro da pterocarpanoquinona LQB 118 e estudo de alguns mecanismos de ação

Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata.

Fundamental limits on the suppression of molecular fluctuations.

Negative feedback is common in biological processes and can increase a system's stability to internal and external perturbations. But at the molecular level, control loops always involve signalling steps with finite rates for random births and deaths of individual molecules. Here we show, by developing mathematical tools that merge control and information theory with physical chemistry, that seemingly mild constraints on these rates place severe limits on the ability to suppress molecular fluctuations. Specifically, the minimum standard deviation in abundances decreases with the quartic root of the number of signalling events, making it extremely expensive to increase accuracy. Our results are formulated in terms of experimental observables, and existing data show that cells use brute force when noise suppression is essential; for example, regulatory genes are transcribed tens of thousands of times per cell cycle. The theory challenges conventional beliefs about biochemical accuracy and presents an approach to the rigorous analysis of poorly characterized biological systems.

一种小G蛋白TaRAN1在植物细胞周期和发育过程中的功能研究

　　小G蛋白作为信号转导中重要的分子开关， 进化相当保守，与许多不同的调控因子和效应器分子相互作用，产生细胞功能的多样性。近年来，人们不断发现植物中小G蛋白家族的新成员，也不断揭示小G蛋白的新功能，许多植物特有的信号途径和功能需要小G蛋白这个重要的分子开关来完成，使它越来越成为人们研究的热点问题。但是，有关植物中Ran GTPase及其编码基因的研究工作报道很少，对与之相互作用的调控蛋白研究进展也刚刚开始。 　　TaRAN1 (AF488730) 是小麦来源的Ran同源蛋白编码基因，全长1055 bp, 编码221个氨基酸，它在植物发育过程中的功能还没有任何报道。本论文在验证了它是小G蛋白Ran家族的成员后，从分子水平上还发现它在植物细胞周期调控、对生长素以及胁迫应答信号转导过程中都起着重要作用，这也说明了它可能作为信号转导过程中重要的转换因子，参与了很多细胞的基本生理过程。 　　利用原核表达系统及亲和色谱的方法纯化了TaRAN1融合蛋白，并用放射性标记的GTP和竞争实验证实了它具有特异的GTP结合活性。TaRAN1的转录产物在小麦幼茎和花芽等分生组织活动旺盛的器官表达较多，而在老叶中表达较少。利用洋葱表皮瞬时表达系统分析表现，TaRAN1蛋白主要定位于细胞核，但其没有典型的核定位信号。 　　细胞周期一直是生物学领域中的热门问题，人们虽然在动物细胞中取得了很大进展，但在植物细胞中的研究远落后于动物。裂殖酵母（Schizosaccharomyces pombe）是研究细胞形态和细胞周期的良好系统，利用此系统发现超表达TaRAN1的酵母细胞表现出许多新的细胞学表型，例如G2细胞周期延滞、染色体对紫外线敏感、细胞超长或多隔细胞的出现等;反义表达TaRAN1的酵母细胞呈近圆型、具有高度凝集的核并且生长速度缓慢、核质混合和无核细胞的数目明显增加。流式细胞仪检测实验也证实其细胞周期的异常。这些结果推测TaRAN1蛋白可能参与细胞周期的有丝分裂过程和发育的调控机制，并且在维持染色体结构稳定和完整性方面起着重要的作用。通过免疫荧光实验观察表明，超表达转基因酵母的微管多呈异常的狭小扇形结构，反义表达TaRAN1的酵母微管不能形成丝状结构，推测TaRAN1还可能参与微管（包括纺锤体）的结构形成过程。最后，我们用超表达TaRAN1的转基因拟南芥和水稻也证实了它的功能，其生长点表现出分生组织增多的原基、根生长点的有丝分裂指数有所改变、出现异常的细胞分裂时相等有关细胞周期异常的现象，更进一步说明了TaRAN1确实参与着细胞周期的调控过程，推测其与细胞周期从G2期进入M期的过程有关。 　　TaRAN1基因受IAA的诱导表达，且随着浓度的增加表达量增强。超表达的TaRAN1植株（包括拟南芥和水稻）的根表现出对外源生长素异常敏感，侧根显著变少，地上部分表现出生长素过量的表现型，顶端优势减弱，分蘖增多，生长周期延长等。HPLC测定转基因植物的IAA含量，明显高于对照。所以，TaRAN1可能还参与了复杂的生长素信号转导过程。TaRAN1基因还受各种胁迫处理的诱导表达，并且超表达植株对胁迫的忍受能力有明显提高，这说明TaRAN1还参与了胁迫信号应答的相应机制。Ran蛋白这些新功能目前还未见到其它报道。

水稻OsMYB3R-2 基因对逆境响应的功能研究

低温威胁水稻的生产，其中苗期和生殖阶段对寒害是最敏感的时期。在苗期，阶段性冷害使水稻幼苗生长延迟，甚至造成烂秧现象；在生殖阶段，无法预测的突然降温会导致水稻花粉不育，并致使水稻大幅减产。因此，对水稻逆境胁迫调控的分子机制的深入研究在理论和实践上具有重要的意义。本研究从东乡野生稻、栽培稻及其杂交后代的低温芯片中筛选对低温响应基因的分析着手，对其中一个受低温诱导上调的基因OsMYB3R-2 作进一步研究。生物信息学的分析表明OsMYB3R-2 编码一个R1R2R3 MYB 蛋白，利用基因枪瞬时转化法、酵母GAL4 系统和电泳迁移率变动分析发现OsMYB3R-2 蛋白能够定位在细胞核中、具有转录激活和DNA 结合特性，表现为MYB 转录因子的典型特征。 超表达OsMYB3R-2 的转基因水稻呈现幼苗的矮化和生长相对滞后的表型，对低温胁迫具有耐受性。盐抑制水稻种子的萌发，与野生型和反义的株系相比，OsMYB3R-2 超表达株系的萌发对盐敏感，表现为萌发过程及萌发之后幼苗的生长更加滞后。而OsMYB3R-2 转基因株系对干旱处理敏感。为了进一步寻找OsMYB3R-2 蛋白的靶序列及其调控的靶基因，我们利用电泳迁移率变动分析发现OsMYB3R-2 能够与有丝分裂特异的激活子（mitosis-specific activator）元件特异结合。在低温条件下，OsMYB3R-2 超表达能够激活水稻G2/M 期特异基因的表达，主要包括OsCycB1;1、OsCycB2;1、OsCycB2;2 和OsCDC20.1 等。另一方面，OsMYB3R-2 超表达能够增加根尖细胞的有丝分裂指数，这进一步说明OsMYB3R-2 参与了水稻细胞周期调控。EMSA、RT-PCR 和流式细胞仪分析的结果表明OsMYB3R-2 通过激活其靶基因OsCycB1;1 的表达参与水稻对低温胁迫的调控，该过程由细胞周期介导。 为了研究OsMYB3R-2 与水稻DREB/CBF 途径的关系，我们分析了转基因水稻中DREB/CBF 类基因及其可能调控的下游基因与OsMYB3R-2 的关系，RT-PCR 的结果表明超表达转基因植物中DREB 表达未见明显变化，而其下游基因OsCPT1 在低温条件下被激活表达。同时，转基因植物在低温条件下脯氨酸水平显著提高。这说明OsMYB3R-2 可能在水稻DREB/CBF 途径的下游参与调控。 总之，OsMYB3R-2 基因的超表达赋予转基因水稻在苗期对低温胁迫具有耐受性，并呈现矮化和生长滞后的表型。OsMYB3R-2 蛋白行使R1R2R3 MYB 转录因子的功能，在体外能够结合OsCycB1;1 和OsKNOLLE2 基因启动子中有丝分裂特异的激活子元件，在低温条件下激活了G2/M 期特异基因的表达，这些基因包括OsCycB1;1、OsCycB2;1、OsCycB2;2 和OsCDC20.1。低温条件下，在OsMYB3R-2 转基因超表达株系中OsCPT1 基因的转录被激活，细胞的游离脯氨酸的含量也显著增高。这些结果都表明OsMYB3R-2 基因在水稻的冷胁迫信号途径中起重要的作用，该过程受细胞周期及DREB/CBF 途径介导。 我们的实验结果暗示水稻对低温的耐受是通过分生组织细胞周期调控完成的，这个过程由OsMYB3R-2 等关键基因控制。

Differential selection on gene translation efficiency between the filamentous fungus Ashbya gossypii and yeasts

Background: The filamentous fungus Ashbya gossypii grows into a multicellular mycelium that is distinct from the unicellular morphology of its closely related yeast species. It has been proposed that genes important for cell cycle regulation play central

Bayesian correlated clustering to integrate multiple datasets.

MOTIVATION: The integration of multiple datasets remains a key challenge in systems biology and genomic medicine. Modern high-throughput technologies generate a broad array of different data types, providing distinct-but often complementary-information. We present a Bayesian method for the unsupervised integrative modelling of multiple datasets, which we refer to as MDI (Multiple Dataset Integration). MDI can integrate information from a wide range of different datasets and data types simultaneously (including the ability to model time series data explicitly using Gaussian processes). Each dataset is modelled using a Dirichlet-multinomial allocation (DMA) mixture model, with dependencies between these models captured through parameters that describe the agreement among the datasets. RESULTS: Using a set of six artificially constructed time series datasets, we show that MDI is able to integrate a significant number of datasets simultaneously, and that it successfully captures the underlying structural similarity between the datasets. We also analyse a variety of real Saccharomyces cerevisiae datasets. In the two-dataset case, we show that MDI's performance is comparable with the present state-of-the-art. We then move beyond the capabilities of current approaches and integrate gene expression, chromatin immunoprecipitation-chip and protein-protein interaction data, to identify a set of protein complexes for which genes are co-regulated during the cell cycle. Comparisons to other unsupervised data integration techniques-as well as to non-integrative approaches-demonstrate that MDI is competitive, while also providing information that would be difficult or impossible to extract using other methods.

Identification of Differentially Expressed Genes Between Cloned and Zygote-Developing Zebrafish (Danio rerio) Embryos at the Dome Stage Using Suppression Subtractive Hybridization

Comparative analyses of differentially expressed genes between somatic cell nuclear transfer (SCNT) embryos and zygote-developing (ZD) embryos are important for understanding the molecular mechanism underlying the reprogramming processes. Herein, we used the suppression subtractive hybridization approach and from more than 2900 clones identified 96 differentially expressed genes between the SCNT and ZD embryos at the dome stage in zebrafish. We report the first database of differentially expressed genes in zebrafish SCNT embryos. Collectively, our findings demonstrate that zebrafish SCNT embryos undergo significant reprogramming processes during the dome stage. However, most differentially expressed genes are down-regulated in SCNT embryos, indicating failure of reprogramming. Based on Ensembl description and Gene Ontology Consortium annotation, the problems of reprogramming at the dome stage may occur during nuclear remodeling, translation initiation, and regulation of the cell cycle. The importance of regulation from recipient oocytes in cloning should not be underestimated in zebrafish.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Gene expression profiles in liver of zebrafish treated with microcystin-LR

Microcystin-LR (MC-LR) is the most frequently studied cyclic heptatoxin produced by cyanobacteria, which has tremendous negative impacts on fish, while its molecular mechanism behind remained unclear at present. Here, Affymetrix Zebrafish GeneChip was used to identify alterations in gene expression of zebrafish (Danio rerio) after MC-LR exposure. Among the 14,900 transcripts in the microarray, 273 genes were differentially expressed, in which 243 genes were elevated and 30 were decreased. According to GOstat analysis, MC-LR mainly influenced the cell cycle and mitogen-activated protein kinases (MAPK) signaling pathways. In addition, many immune-related genes were also influenced. These data suggest that MC-LR could promote tumorigenesis and cause immunotoxicity in fish. (C) 2008 Elsevier B.V. All rights reserved.

Constitutive expression of thymidylate synthase from LCDV-C induces a transformed phenotype in fish cells

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumor formation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virus from China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4-5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity. (C) 2007 Elsevier Inc. All rights reserved.

Identification of differentially expressed genes from the cross-subfamily cloned embryos derived from zebrafish nuclei and rare minnow enucleated eggs

Cross-species nuclear transfer (NT) has been used to retain the genetic viability of a species near extinction. However, unlike intra-species NT, most embryos produced by cross-species NT were unable to develop to later stages due to incompatible nucleocytoplasmic interactions between the donor nuclei and the recipient cytoplasm from different species. To study the early nucleocytoplasmic interaction in cross-species NT, two laboratory fish species (zebrafish and rare minnow) from different subfamilies were used to generate cross-subfamily NT embryos in the present study. Suppression subtractive hybridization (SSH) was performed to screen out differentially expressed genes from the forward and reverse subtractive cDNA libraries. After dot blot and real-time PCR analysis, 80 of 500 randomly selective sequences were proven to be differentially expressed in the cloned embryos. Among them, 45 sequences shared high homology with 28 zebrafish known genes, and 35 sequences were corresponding to 22 novel expressed sequence tags (ESTs). Based on functional clustering and literature mining analysis, up-and down-regulated genes in the cross-subfamily cloned embryos were mostly relevant to transcription and translation initiation, cell cycle regulation, protein binding, etc. To our knowledge, this is the first report on the determination of genes involved in the early development of cross-species NT embryos of fish. (C) 2007 Elsevier Inc. All rights reserved.

Apoptosis induction on human hepatoma cells Hep G2 of decabrominated diphenyl ether (PBDE-209)

Polybrominated diphenyl ethers (PBDEs) are an important class of halogenated organic brominated flame retardants. Because of their presence in abiotic and biotic environments widely and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible adverse health effects to humans. This study was designed to determine the anti-proliferative, apoptotic properties of decabrominated diphenyl ether (PBDE-209), using a human hepatoma Hep G2 line as a model system. Hep G2 cells were cultured in the presence of PBDE-209 at various concentrations (1.0-100.0 mu mol/L) for 72 h and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that PBDE-209 inhibited the cells viability in time and concentration-dependent characteristics at concentrations (10.0-100.0 mu mol/L). We found that anti-proliferative effect of PBDE-209 was associated with apoptosis on Hep G2 cells by determinations of morphological changes, cell cycle and apoptosis. Mechanism study showed that PBDE-209 could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. The mechanism for its hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation. In addition, activity of lactate dehydrogenase (LDH) release increased when the cells incubated with PBDE-209 at various concentrations and times. These results suggested that PBDE-209 had the toxicity activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.