Nucleosomes represent a physical barrier for cleavage activity of DNA topoisomerase I in vivo

DNA topoisomerase I together with the other cellular DNA topoisomerases releases the torsional stress from DNA caused by processes such as replication, transcription and recombination. Despite the well-defined knowledge of its mechanism of action, DNA topoisomerase I in vivo activity has been only partially characterized. In fact the basic question concerning the capability of the enzyme to cleave and rejoin DNA wrapped around a histone octamer remains still unanswered. By studying both in vivo and in vitro the cleavage activity of DNA topoisomerase I in the presence of camptothecin on a repeated trinucleotide sequence, (TTA)35, lying in chromosome XIII of Saccharomyces cerevisiae, we can conclude that nucleosomes represent a physical barrier for the enzyme activity. © The Authors

FOB1 affects DNA topoisomerase I in vivo cleavages in the enhancer region of the Saccharomyces cerevisiae ribosomal DNA locus

In Saccharomyces cerevisiae the FOB1 gene affects replication fork blocking activity at the replication fork block (RFB) sequences and promotes recombination events within the rDNA cluster. Using in vivo footprinting assays we mapped two in vivo Fob1p-binding sites, RFB1 and RFB3, located in the rDNA enhancer region and coincident with those previously reported to be in vitro binding sites. We previously provided evidences that DNA topoisomerase I is able to cleave two sites within this region. The results reported in this paper, indicate that the DNA topoisomerase I cleavage specific activity at the enhancer region is affected by the presence of Fob1p and independent of replication and transcription activities. We thus hypothesize that the binding to DNA of Fob1p itself may be the cause of the DNA topoisomerase I activity in the rDNA enhancer

Effect of Aminaphtone on in vitro vascular permeability and capillary–like maintenance

Aminaphtone (AMNA), a naphtohydrochinone used in the treatment of capillary disorders, may affect oedema in chronic venous insufficiency (CVI). Aim of study is to investigate the effect of AMNA on vascular endothelial permeability in vitro and its effects on three-dimensional capillary-like structures formed by Human Umbilical Vein Endothelial Cells (HUVECs). HUVECs were treated with 50 ng/ml VEGF for 2h and AMNA for 6h. Permeability assay, VE-cadherin expression and Matrigel assay were performed. VEGF-induced permeability was significantly decreased by AMNA in a range concentration of 1-20 μg/ml. AMNA restored VE-cadherin expression. Finally, 6h pretreatment with AMNA significantly preserved capillary-like structures formed by HUVECs on Matrigel up to 48 h compared to untreated cells. AMNA significantly protects endothelium permeability and stabilises endothelial cells organised in capillary-like structures, modulating VE-cadherin expression. These data might explain the clinical benefict of AMNA on CVI

Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes

Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation. Our findings demonstrate that residues 115–125 within the yeast Top1p N-terminal domain are required for the complementation of the top1Δ rDNA phenotypes. In chromatin immunoprecipitation and co-immunoprecipitation experiments, we further demonstrate the physical interaction between Top1p and Sir2p. Our genetic and biochemical studies support a model whereby Top1p recruits Sir2p to the rDNA and clarifies a structural role of DNA topoisomerase I in the epigenetic regulation of rDNA, independent of its known catalytic activity

What is a Gene? A Two Sided View

The need to account for all currently available experimental observations concerning the gene nature, has reshaped the concept of gene turning it from the essentially mechanistic unit, predominant during the '70s, into a quite abstract open and generalized entity, whose contour appears less defined as compared to the past. Here we propose the essence of the gene to be considered double faced. In this respect genotypic and phenotypic entities of a gene would coexist and mix reciprocally. This harmonizes present knowledge with current definitions and predisposes for remodelling of our thinking as a consequence of future discoveries. A two sided view of the gene also allows to combine the genetic and epigenetic aspects in a unique solution, being structural and functional at the same time and simultaneously able to include the different levels in an overlapping unicum

Inclusive Physical Activity to Promote the Participation of People with Disabilities: A Preliminary Study

Background: Physical activity brings improvements in the quality of life in all individuals, disabled and non-disabled. There is little evidence in the literature of inclusive physical activity in which disabled and non-disabled people participate at the same level. Objectives: The study aimed to demonstrate the effectiveness of an inclusive training program, structured in such a way as to encourage physical activity for all participants with and without disabilities, in improving body composition, explosive strength, and endurance. Methods: A sample of twenty-four subjects (mean age: 24.09±3.92 years), 12 disabled and 12 non-disabled, was selected. Quantitative input and output data were recruited at 16-week intervals using a battery of tests: anthropometric measurements, Vertec Squat Jump test, and Yoyo Endurance Test. During the 16 weeks, all participants followed an appropriately structured training program in four mesocycle without any differences. Input and output data were compared employing the t-test for dependent samples. Results and conclusions: The results showed statistically meaningful improvements at an alpha level set at 0.05 for the three parameters tested. These results confirmed the effectiveness of the proposed inclusive training protocol on the improvement of the tested parameters in all participants. These strategies didn't jeopardise the achievement of the overall objectives set; on the contrary, improvements in BMI, explosive strength, and endurance strength of 4.8%, 4.3%, and 56.2% respectively were observed