Development and Characterization of cDNA Resources for the Common Marmoset: One of the Experimental Primate Models

The common marmoset is a new world monkey, which has become a valuable experimental animal for biomedical research. This study developed cDNA libraries for the common marmoset from five different tissues. A total of 290 426 high-quality EST sequences were obtained, where 251 587 sequences (86.5%) had homology (1E⁻¹⁰⁰) with the Refseqs of six different primate species, including human and marmoset. In parallel, 270 673 sequences (93.2%) were aligned to the human genome. When 247 090 sequences were assembled into 17 232 contigs, most of the sequences (218 857 or 15 089 contigs) were located in exonic regions, indicating that these genes are expressed in human and marmoset. The other 5578 sequences (or 808 contigs) mapping to the human genome were not located in exonic regions, suggesting that they are not expressed in human. Furthermore, a different set of 118 potential coding sequences were not similar to any Refseqs in any species, and, thus, may represent unknown genes. The cDNA libraries developed in this study are available through RIKEN Bio Resource Center. A Web server for the marmoset cDNAs is available at http://marmoset.nig.ac.jp/index.html, where each marmoset EST sequence has been annotated by reference to the human genome. These new libraries will be a useful genetic resource to facilitate research in the common marmoset.     

Analysis of waving and sound‐production display in the ghost crab,Ocypode stimpsoni

The ghost crab Ocypode stimpsoni displays waving and sound production. Sounds are produced by thumping the sand substratum with the major cheliped, and two types of sounds can be discriminated; one with a low frequency of about 12 Hz, called rapping, and another with a higher frequency (about double), called quivering. In our observations, a sequence of waving and sound emission would sometimes terminate abruptly, or appear as independent components but the component order never changed. The most frequently observed patterns were “waving with rapping and quivering”;, “waving with quivering”; and “quivering only”; quivering sounds being involved in more than 80% displays. Quivering sometimes occurred immediately after crabs emerged from the burrow, or when they returned to the entrance after discarding an excavated sand mass. The occurrence frequency of waving and sounds, the wave amplitude, and the frequency of the sound increased when other crabs approached.     

Influence of drying methods over in vitro antitumoral effects of exopolysaccharides produced by Agaricus blazei LPB 03 on submerged fermentation

Agaricus blazei is a mushroom that belongs to the Brazilian biodiversity and is considered as an important producer of bioactive compounds beneficial to human health. Studies have demonstrated that these compounds present immuno-modulatory, antioxidant and antitumor properties. In order to compare the most used method for fungal polysaccharide drying, lyophilization with other industrial-scale methods, the aim of this work was to submit A. blazei LPB 03 polysaccharide extracts to vaucum, spray and freeze drying, and evaluate the maintenance of its antitumoral effects in vitro. Exopolysaccharides produced by A. blazei LPB 03 on submerged fermentation were extracted with ethanol and submitted to drying processes. The efficiency represents the water content that was removed during the drying process. The resultant dried products showed water content around 3% and water activity less than 0.380, preventing therefore the growth of microorganisms and reactions of chemical degradation. Exopolysaccharide extracts dried by vacuum and spray dryer did not showed any significant cytotoxic effect on cell viability of Wistar mice macrophages. Content of total sugars and protein decrease after drying, nevertheless, 20 mg/ml of exopolysaccharides dried by spray dryer reached 33% of inhibition rate over Ehrlich tumor cells in vitro.     

Finding complexity in complexes: Assessing the causes of mitonuclear discordance in a problematic species complex of Mesoamerican toads

Mitonuclear discordance is a frequently encountered pattern in phylogeographic studies and occurs when mitochondrial and nuclear DNA display conflicting signals. Discordance among these genetic markers can be caused by several factors including confounded taxonomies, gene flow, and incomplete lineage sorting. In this study, we present a strong case of mitonuclear discordance in a species complex of toads (Bufonidae: Incilius coccifer complex) found in the Chortís Block of Central America. To determine the cause of mitonuclear discordance in this complex, we used spatially explicit genetic data to test species limits and relationships, characterize demographic history, and quantify gene flow. We found extensive mitonuclear discordance among the three recognized species within this group, especially in populations within the Chortís Highlands of Honduras. Our data reveal nuclear introgression within the Chortís Highlands populations that was most probably driven by cyclical range expansions due to climatic fluctuations. Though we determined introgression occurred within the nuclear genome, our data suggest that it is not the key factor in driving mitonuclear discordance in the entire species complex. Rather, due to a lack of discernible geographic pattern between mitochondrial and nuclear DNA, as well as a relatively recent divergence time of this complex, we concluded that mitonuclear discordance has been caused by incomplete lineage sorting. Our study provides a framework to test sources of mitonuclear discordance and highlights the importance of using multiple marker types to test species boundaries in cryptic species.     

Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression

We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern for some C/D box snoRNAs and abundant expression of longer, non-coding RNAs in the cell that can potentially form dsRNAs.     

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell–cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell–cell and/or cell–ECM contacts and anchorage-independent cell proliferation.     

Genistein, isoflavonoids in soybeans, prevents the formation of excess radiation-induced centrosomes via p21 up-regulation

The centrosome is a cytoplasmic organelle which duplicates once during each cell cycle, and the presence of excess centrosomes promote chromosome instability through chromosome missegregation following cytokinesis. Ionizing radiation (IR) can induce extra centrosomes by permitting the continuation of CDK2/Cyclin-A/E-mediated centrosome duplication when cells are arrested in the cell cycle after irradiation. The work described here shows that, in addition to IR, extra centrosomes were induced in human U2OS and mouse NIH3T3 cells after treatment with agents which include DNA adduct-forming chemicals: benzopyrene (BP), 4-nitroquinoline 1-oxide (4NQO), a DNA cross linker: cis-diamminedichloro-platinum (cisplatin), topoisomerase inhibitors: camptothecin, etoposide, genistein, and ultra-violet light (UV). These agents were divided into two categories with respect to the regulation of p21, which is an inhibitor of CDK2/Cyclin-A/E: specifically, p21 was up-regulated by an IR exposure and treatment with topoisomerase inhibitors. However, UV, BP, 4NQO and cisplatin down-regulated p21 below basal levels. When cells were irradiated with IR in combination with all of these agents, except genistein, enhanced induction of extra centrosomes was observed, regardless of the nature of p21 expression. Genistein significantly suppressed the frequency of IR-induced extra centrosomes in a dose-dependent manner, and 20μg/ml of genistein reduced this frequency to 66%. Consistent with this, genistein substantially up-regulated p21 expression over the induction caused by IR alone, while other agents down-regulated or marginally affected this. This suggests the inhibitory effect of genistein on the induction of extra centrosomes occurs through the inactivation of CDK2/Cyclin-A/E via p21 up-regulation. This hypothesis is supported by the observation that p21 knockdown with siRNA reduced the activity of CDK2/Cyclin-A/E and restored the enhanced effect of a combined treatment with genistein and IR. These results demonstrate the preventive effect of genistein and a crucial role for p21 in IR-induced excess centrosomes.