Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting thatXIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance ofXIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.

Possible consequences of increasing life expectancy in Brazil: the perspective of a European historical demographer

Those over sixty years of age accounted for 6.6% of the total population of Brazil in 1985, in the Federal Republic of Germany this proportion was 20.3% in 1984. As early as 1950 it had been 14.5%. This proportion will not even be reached in Brazil in the year 2000 when persons aged sixty years and older are only projected to make up 8.8% of the total population. Similarly, in 1982/84 life expectancy at birth in the Federal Republic was 70.8 years for men and 77.5 for women; in Brazil the figures for 1980/85 were, by contrast, "only" 61.0 and 66.0. Against this background it is easy to understand why the discussion concerning an ageing society with its many related medical, economic, individual and social problems has been so slow in coming into its own in Brazil. As important as a more intensive consideration of these aspects may be in Brazil at present, they are, nevertheless, only one side of the story. For a European historical demographer with a long-term perspective of three of four hundred years, the other side of the story is just as important. The life expectancy which is almost ten years lower in Brazil is not a result of the fact that no one in Brazil lives to old age. In 1981 people sixty-five years and older accounted for 34.4% of all deaths! At the same time infants accounted for only 22.1% of total mortality. They are responsible, along with the "premature" deaths among youths and adults, for the low, "average" life expectancy figure. In Europe, by contrast, these "premature" deaths no longer play much of a role. In 1982/84 more than half of the women (52.8%) in the Federal Republic of Germany lived to see their eightieth birthdays and almost half of the men (47.3%) lived to see their seventy-fifth. Our biological existence is guaranteed to an extent today that would have been unthinkable a few generations ago. Then, the classic troika of "plague, hunger and war" threatened our forefathers all the time and everywhere. The radical transition from the formerly uncertain to a present-day certain lifetime, which is the result of the repression of "plague, hunger and war", led to unexpected consequences for our living together. Our forefathers were forced to live in closely knit Gemeinschaften in the interest of physical survival and to subordinate their egoistic goals to a common value, but now these pressures have, for the most part, fallen away. Correspondingly, this much more certain EGO has taken center stage. An ever greater number of us chooses to live life as single beings: the number of marriages is lower every year; the number of divorces is on the increase; in Berlin (West) more than half (sic! 52.3%) of all households are already composed on only one person. For the last dozen years the annual number of births in the Federal Republic has been insufficient to ensure population replacement. Not a population explosion but rather the opposite, a population implosion, is our problem. Human beings do not appear to be "social animals", as was axiomatically assumed for so long. They were only forced to behave as such for as long as "plague, hunger and war" forced them to do so. When these life endangering conditions no longer exist and life becomes certain even without their being integrated into a Gemeinschaft then humans suddenly show themselves more and more to be independent single beings. It is not the percentage of the population that is over sixty or sixty-five that is decisive in this context but rather how certain adults perceive their biological lives to be, since they are the ones who organize their lives, who build communities or who are ever more often willing only to enter into means-to-an-end personal unions without lasting or close ties and mutual responsibilities. There are many signs which seem to point to a development in this direction in Brazil as well. More and more adults in Brazil are caught up in the deep-seated transition from an uncertain to a certain lifetime. A third of them die after having reached their sixty-fifth birthday. It therefore seems to me to be high time that one began to give more consideration to the other side of the story in Brazil as well. And who is more suited intensively to consider the long-term perspectives than those engaged in the public health sector in whose competence, after all, such aspects, as "life certainty", "life expectancy" and "age at death" belong?

Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

Microcalorimetria: uma técnica aplicável ao estudo do diauxismo da Saccharomyces cerevisiae

In this work we first introduce the reader to the basic concepts of biology, bioenergetics and biochemistry, concerning the area of cell biology. Then we explain what diauxism is and an example of this phenomenon, applied to S. cerevisiae, is presented. Finally, thermograms obtained by microcalorimetry, from S. cerevisiae that undergo diauxism, are discussed from a biochemical point of view.

miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukemia

MicroRNAs (miRNAs) are a class of small endogenous RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression. miRNAs act in diverse biological processes including development, cell growth, apoptosis, and hematopoiesis, suggesting their association with cancer. We determined the miRNA expression profile of chronic and acute lymphocytic leukemias (CLL and ALL) using the TaqMan® MicroRNA Assays Human Panel (Applied Biosystems). Pooled leukemia samples were compared to pooled CD19+ samples from healthy individuals (calibrator) by the 2-DDCt method. Total RNA input was normalized based on the Ct values obtained for hsa-miR-30b. The five most highly expressed miRNAs were miR-128b, miR-204, miR-218, miR-331, and miR-181b-1 in ALL, and miR-331, miR-29a, miR-195, miR-34a, and miR-29c in CLL. To our knowledge, this is the first report associating miR-128b, miR-204 and miR-331 to hematological malignancies. The miR-17-92 cluster was also found to be up-regulated in ALL, as previously reported for some types of lymphomas. The differences observed in gene expression levels were validated for miR-331 and miR-128b in ALL and CD19+ samples. These miRNAs were up-regulated in ALL, in agreement with our initial results. A brief target analysis was performed for miR-331. One of its putative targets, SOCS1, promotes STAT activation, which is a known mediator of cell proliferation and survival, suggesting the possibility of an association between miR-331 and these processes. This initial screening provided information on miRNA differentially expressed in normal and malignant B-cells that could suggest the potential roles of these miRNAs in hematopoiesis and leukemogenesis.

The miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation of mouse embryonic cardiomyocytes

MicroRNAs (miRNAs) have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92) cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2) is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation in mouse embryonic cardiomyocytes we initially used bioinformatics to analyze 3’ untranslated regions (3’UTR) of FOG-2 to predict the potential of miR-17-92 to target it. We used luciferase assays to demonstrate that miR-17-5p and miR-20a of miR-17-92 interact with the predicted target sites in the 3’UTR of FOG-2. Furthermore, RT-PCR and Western blot were used to demonstrate the post-transcriptional regulation of FOG-2 by miR-17-92 in embryonic cardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdU cell assays together with the FOG-2 rescue strategy were employed to evaluate the effect of proliferation on embryonic cardiomyocytes. We first found that the miR-17-5p and miR-20a of miR-17-92 directly target the 3’UTR of FOG-2 and post-transcriptionally repress the expression of FOG-2. Moreover, our findings demonstrated that over-expression of miR-17-92 may inhibit cell proliferation via post-transcriptional repression of FOG-2 in embryonic cardiomyocytes. These results indicate that the miR-17-92 cluster regulates the expression of FOG-2 protein and suggest that the miR-17-92 cluster might play an important role in heart development.

The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C2H2 zinc finger protein

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C2H2 motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

Assessing development and the idea of development in the 1950s in Brazil

The decade of 1950s was a crucial period of the industrialization of the Brazilian economy. The dominant school of thought was the national-developmentalism, which was not restricted to the sphere of economic production but also encompassed political and socio-cultural processes of change. Combining repression, persuasion and paternalism, the national state took a significantly political and economic responsibility in the social, material and symbolic modernization during the Vargas and Kubitschek administrations. However, internal disputes, foreign demands and a long legacy of socio-spatial inequalities prevented the achievement of more socially inclusive goals, leading a legacy of unanswered questions that still have currency today.