Aldina Brás

Oxidative stress in trisomy 21: A possible role in cataractogenesis

Previous studies have suggested that free radicals and related species play a role in lens damage. The molecules involved may include proteins, lipids and DNA. Focal cortical changes and cortical liquefaction have been reported in patients with Downs syndrome over the age of 15 years. There is evidence supporting the hypothesis that trisomy 21 patients have an increase in free radical reactions and lipoperoxidation susceptibility. This could be due to an increase in the H2O2 generation catalysed by CuZn SOD although the activity of other gene products coded for on chromosome 21 cannot be excluded. Thiobarbituric acid reactive products were measured in human erythrocytes of nine DS patients and nine age-matched controls. There was a significant increase in the first group (21.0 +/- 2.3 nmol MDA/g Hb vs 16.4 +/- 2.9 nmol MDA/g Hb; p less than or equal to 0.01). In plasma, however, TBA products and antioxidant levels (ascorbic acid, tocopherol and uric acid) were not significantly different. Further studies should be carried out, namely through the use of more specific and sensitive methods, to assess the possible association between oxidative stress and cortical lens damage in DS patients.

Oxidative stress in familial adenomatous polyposis.

The prooxidant/antioxidant imbalance in familial adenomatous polyposis (FAP) is suggested by (i) the intimate connection between APC and prostaglandin H synthase-2 genes, (ii) the increase of the free radical-generating enzyme xanthine oxidase, and (iii) the decrease of antioxidant defences. In this research work we evaluated lipid peroxidation measuring the thiobarbituric acid (TBA) reactive products and we studied the activities of superoxide dismutase (SOD) and catalase as well as the levels of ascorbate and tocopherols in the peripheral blood cells from a total of 27 FAP patients and 83 normal controls. TBA-reactive products were determined according to a previously published method. SOD and catalase activities were determined by the spectrophotometric monitoring of the inhibition of pyrogallol autoxidation and the hydrogen peroxide decomposition rate, respectively. Ascorbate levels were determined by a modified 2,4-dinitrophenylhydrazine method and tocopherol levels by a modified Emmerie-Engle method. The levels of TBA-reactive products were higher in FAP patients than in normal controls. Although no statistically significant differences in SOD and catalase activities were observed between FAP patients and normal controls, we found that ascorbate and tocopherol levels were significantly lower in FAP patients than in normal controls, as assessed by the Mann-Whitney test. Hence, this finding of an imbalance in the prooxidant/antioxidant status may contribute towards new strategies for prevention and therapy in FAP patients.

DNA Damage and Oxygen Species

Various genes are involved in cell protection against oxidative stress (e.g., SOD, catalase, GSH peroxidase) and some genes recognize and repair oxidative damage to DNA (both base and sugar damage). Yet, the nature and relative rates of the reactions involving active oxygen species make it difficult to ascertain, in each particular situation, what reactants may act as the predominant genotoxicant and what DNA repair mechanisms may ensue.

Telomerase and reactive oxygen species: Comments on Saretzki, G., 2009. Telomerase, mitochondria and oxidative stress. Exp. Gerontol. 44, 485–492

We have read with great interest the article by Gabriele Saretzki and really appreciated the excellent review of the subject ‘‘Telomerase, mitochondria and oxidative stress” as well as the scientific work that this group has been doing. The author points out that the finding of a mitochondria-protective function of telomerase from two labs (Ahmed et al., 2008; Haendeler et al., 2009) directly contradicts the observations of Santos et al. (2004, 2006) who showed an adverse effect of mitochondrial localisation of telomerase on the integrity of mitochondrial DNA leading to apoptosis. However, as pointed by the author, it is not clear by what mechanism hTERT protects mitochondrial DNA (Saretzki, 2009). One of the possible mechanisms suggested by Saretzki is the accelerated degradation of mitochondria harbouring damaged DNA and this may not be inconsistent with the findings published by Santos et al. (2006). Interestingly, Tondera et al. (2009) described a pathway that they call stress-induced mitochondrial hyperfusion (SIMH) pathway, which is induced by low levels of stress. This closed network confers some degree of resistance to further insults, perhaps by increasing the robustness of mitochondria through exchange of crucial components, but eventually cells might succumb to apoptosis (van der Bliek, 2009). In a cellular context, telomerase has many faces, not all of which are fully understood yet (Bollmann, 2008). Not only telomerase is influenced by the level of oxidative stress, but is regulated in a cell-dependent manner (Yang et al., 2008). The levels and localization of ROS are likely to determine whether the ROS serve as adaptive signaling or damaging molecules (Klimova et al., 2009). Translocation of telomerase into the mitochondria upon oxidative stress can occur within less than 3 h under H2O2 treatment which essentially excludes de novo synthesis as the source of the mitochondrial hTERT protein pool (Ahmed et al., 2008; Saretzki, 2009). In our lab, we established cultures of isolated lymphocytes from peripheral blood from a total of 8 healthy individuals, non-smokers, not taking drugs, without recent exposure to radiation and with ages between 19 and 57 years. The principles of the Declaration of Helsinki have been observed. All the individuals gave their informed consent prior to their inclusion in the study. The lymphocytes were separated by the Ficoll-paque method, stimulated with phytohemagglutinin (PHA) 8 lg/ml, and cultured in Ham’s F10 with 24% fetal calf serum (FCS). Recombinant human interleukine-2 (IL-2) was added to cultures on the 3rd day and twice a week since then in order to maintain

Presymptomatic diagnosis in Portuguese FAP families using intragenic RFLPs and (CA)n flanking markers by fluorescence based semiautomated DNA analysis.

Owing to the large size of the APC gene, responsible for familial adenomatous polyposis, direct screening for individual mutations is not a practical approach. In the present study we establish the methodology of fluorescence based semi-automated DNA analysis to perform presymptomatic diagnosis of members at risk from 11 Portuguese FAP families with three (CA)n markers flanking the APC gene, MBC, CB26, and YN5.64, and four intragenic RFLPs. Haplotypes were constructed on the basis of individual genotypes and their segregation through generations were followed. The study was informative for 12% of subjects using only intragenic RFLPs and increased to 90% when we used the three (CA)n flanking markers. We report two of the 11 families under study in our laboratory and show recombinant events leading to a precise localisation of the CB26 marker between D5S82 and the APC gene. In one family there was a loss of (CA) units of one allele of the CB26 marker from an unaffected mother to her son.

Normal Genetic Response To Gamma-irradiation in Familial Adenomatous Polyposis

The present study, a co-operative project between three European institutes, was aimed at elucidating whether the APC gene in carriers of familial adenomatous polyposis coli (FAP) also causes some genetic sensitivity revealed by DNA damage and the yield of chromosome aberrations in peripheral blood lymphocytes exposed to gamma rays. In addition, it seemed of interest to study whether DNA repair is modified after irradiation of lymphocytes from FAP patients compared to controls. To this end, we have used the inhibition of the poly(ADP-ribose) polymerase (ADPRP) by 3-aminobenzamide (3ABA) and studied the effect of 3ABA on the frequency of DNA strand breaks and chromosome aberrations. The data indicate that FAP is not associated with an increased chromosomal sensitivity towards ionising radiation.

Down syndrome and microRNAs (Review)

In recent years numerous studies have indicated the importance of microRNAs (miRNA/miRs) in human pathology. Down syndrome (DS) is the most prevalent survivable chromosomal disorder and is attributed to trisomy 21 and the subsequent alteration of the dosage of genes located on this chromosome. A number of miRNAs are overexpressed in down syndrome, including miR-155, miR-802, miR- 125b-2, let-7c and miR-99a. This overexpression may contribute to the neuropathology, congenital heart defects, leukemia and low rate of solid tumor development observed in patients with DS. MiRNAs located on other chromosomes and with associated target genes on or off chromosome 21 may also be involved in the DS phenotype. In the present review, an overview of miRNAs and the haploinsufficiency and protein translation of specific miRNA targets in DS are discussed. This aimed to aid understanding of the pathogenesis of DS, and may contribute to the development of novel strategies for the prevention and treatment of the pathologies of DS.

The human chromosomal fragile sites more often involved in constitutional deletions and duplications – A genetic and statistical assessment

Human chromosomal fragile sites (CFSs) are heritable loci or regions of the human chromosomes prone to exhibit gaps, breaks and rearrangements. Determining the frequency of deletions and duplications in CFSs may contribute to explain the occurrence of human disease due to those rearrangements. In this study we analyzed the frequency of deletions and duplications in each human CFS. Statistical methods, namely data display, descriptive statistics and linear regression analysis were applied to analyze this dataset. We found that FRA15C, FRA16A and FRAXB are the most frequently involved CFSs in deletions and duplications occurring in the human genome.

Integration of HIV in the Human Genome: Which Sites Are Preferential? A Genetic and Statistical Assessment

Chromosomal fragile sites (FSs) are loci where gaps and breaks may occur and are preferential integration targets for some viruses, for example, Hepatitis B, Epstein-Barr virus, HPV16, HPV18, and MLV vectors. However, the integration of the human immunodeficiency virus (HIV) in Giemsa bands and in FSs is not yet completely clear. This study aimed to assess the integration preferences of HIV in FSs and in Giemsa bands using an in silico study. HIV integration positions from Jurkat cells were used and two nonparametric tests were applied to compare HIV integration in dark versus light bands and in FS versus non-FS (NFSs). The results show that light bands are preferential targets for integration of HIV-1 in Jurkat cells and also that it integrates with equal intensity in FSs and in NFSs. The data indicates that HIV displays different preferences for FSs compared to other viruses. The aim was to develop and apply an approach to predict the conditions and constraints of HIV insertion in the human genome which seems to adequately complement empirical data.

Genetic and Statistical Study of HIV Integration in the Human Genome

Integration of the human immunodeficiency virus (HIV) DNA into human genome is essential for HIV-induced disease. The human genome is organized into chromosomes and within these we can define the chromosomal fragile sites. Our aim is to contribute to help clarifying the integration sites preferences of HIV1 and HIV2 in fragile or non-fragile regions. Here we apply statistical techniques, namely non-parametric tests and analysis of variance for analyzing two sets of data of HIV1 and HIV2 integrations in the human genome. The results show that the integrations occur significantly with more intensity in the non-fragile regions of the human genome and that the HIV1 in particular has the major contribution to this fact. This study could have implications in human disease.