Polymorphisms -455G/A and -148C/T and Fibrinogen Plasmatic Level as Risk Markers of Coronary Disease and Major Adverse Cardiovascular Events.

Some polymorphisms in genes codifying for fibrinogen have been correlated with plasma levels of this protein, and several studies reported their associations with acute cardiovascular events. In the present study, 118 subjects with unstable and stable coronary diseases were enrolled to determinate the associations among fibrinogen gene polymorphisms, plasma fibrinogen levels, and major cardiovascular adverse events in a sample of southwestern Mexico. The groups, including 81 control subjects, were matched for age, sex, body mass index, and sedentarism. Plasma fibrinogen levels and the polymorphisms 455G/A, -148C/T, +1689T/G, and Bcl I of the gene of fibrinogen were compared in all groups. Plasma fibrinogen levels (>465 mg/dl) were significant in patients with coronary disease. Fibrinogen plasma values > 450 mg/dl were associated with cardiovascular mortality during the follow-up analysis of the unstable coronary disease group (p = 0.04). The allelic loads of -455A and -148T were associated with plasma fibrinogen levels > 450 mg/dl (p < 0.003 and p = 0.03, respectively) and with coronary disease (p = 0.016 and p < 0.006, respectively). The follow-up of posterior events after an acute coronary event showed that the genetic load of the -148T allele was associated with major adverse cardiovascular events (RR = 1.8, 95%CI = 1.01-3.35, p = 0.04). Fibrinogen plasmatic levels > 450 mg/dl and the fibrinogen polymorphisms -455G/A and 148C/T had association with MACE and coronary disease. This study suggests that these gene polymorphisms are associated with cardiovascular risk.

Aging and CaMKII alter intracellular Ca2+ transients and heart rhythm in Drosophila melanogaster.

Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility.

Trafficking and chromatin dynamics of holocarboxylase synthetase during development of Drosophila melanogaster.

This work examines the cellular localization of holocarboxylase synthetase (HCS) and its association to chromatin during different stages of development of Drosophila melanogaster. While HCS is well known for its role in the attachment of biotin to biotin-dependent carboxylase, it also regulates the transcription of HCS and carboxylases genes by triggering a cGMP-dependent signal transduction cascade. Further, its presence in the nucleus of cells suggests additional regulatory roles, but the mechanism involved has remained elusive. In this study, we show in D. melanogaster that HCS migrates to the nucleus at the gastrulation stage. In polytene chromosomes, it is associated to heterochromatin bands where it co-localizes with histone 3 trimethylated at lysine 9 (H3K9met3) but not with the euchromatin mark histone 3 acetylated at lysine 9 (H3K9ac). Further, we demonstrate the association of HCS with the hsp70 promoter by immunofluorescence and chromatin immuno-precipitation (ChIP) of associated DNA sequences. We demonstrate the occupancy of HCS to the core promoter region of the transcriptionally inactive hsp70 gene. On heat-shock activation of the hsp70 promoter, HCS is displaced and the promoter region becomes enriched with the TFIIH subunits XPD and XPB and elongating RNA pol II, the latter also demonstrated using ChIP assays. We suggest that HCS may have a role in the repression of gene expression through a mechanism involving its trafficking to the nucleus and interaction with heterochromatic sites coincident with H3K9met3.

p8/TTDA overexpression enhances UV-irradiation resistance and suppresses TFIIH mutations in a Drosophila trichothiodystrophy model.

Mutations in certain subunits of the DNA repair/transcription factor complex TFIIH are linked to the human syndromes xeroderma pigmentosum (XP), Cockayne's syndrome (CS), and trichothiodystrophy (TTD). One of these subunits, p8/TTDA, interacts with p52 and XPD and is important in maintaining TFIIH stability. Drosophila mutants in the p52 (Dmp52) subunit exhibit phenotypic defects similar to those observed in TTD patients with defects in p8/TTDA and XPD, including reduced levels of TFIIH. Here, we demonstrate that several Dmp52 phenotypes, including lethality, developmental defects, and sterility, can be suppressed by p8/TTDA overexpression. TFIIH levels were also recovered in rescued flies. In addition, p8/TTDA overexpression suppressed a lethal allele of the Drosophila XPB homolog. Furthermore, transgenic flies overexpressing p8/TTDA were more resistant to UV irradiation than were wild-type flies, apparently because of enhanced efficiency of cyclobutane-pyrimidine-dimers and 6-4 pyrimidine-pyrimidone photoproducts repair. This study is the first using an intact higher-animal model to show that one subunit mutant can trans-complement another subunit in a multi-subunit complex linked to human diseases.

DNA repair and transcriptional deficiencies caused by mutations in the Drosophila p52 subunit of TFIIH generate developmental defects and chromosome fragility.

The transcription and DNA repair factor TFIIH is composed of 10 subunits. Mutations in the XPB, XPD, and p8 subunits are genetically linked to human diseases, including cancer. However, no reports of mutations in other TFIIH subunits have been reported in higher eukaryotes. Here, we analyze at genetic, molecular, and biochemical levels the Drosophila melanogaster p52 (DMP52) subunit of TFIIH. We found that DMP52 is encoded by the gene marionette in Drosophila and that a defective DMP52 produces UV light-sensitive flies and specific phenotypes during development: organisms are smaller than their wild-type siblings and present tumors and chromosomal instability. The human homologue of DMP52 partially rescues some of these phenotypes. Some of the defects observed in the fly caused by mutations in DMP52 generate trichothiodystrophy and cancer-like phenotypes. Biochemical analysis of DMP52 point mutations introduced in human p52 at positions homologous to those of defects in DMP52 destabilize the interaction between p52 and XPB, another TFIIH subunit, thus compromising the assembly of the complex. This study significantly extends the role of p52 in regulating XPB ATPase activity and, consequently, both its transcriptional and nucleotide excision repair functions.

TFIIH trafficking and its nuclear assembly during early Drosophila embryo development.

We present the first analysis of the dynamics of the transcription DNA-repair factor TFIIH at the onset of transcription in early Drosophila development. TFIIH is composed of ten polypeptides that are part of two complexes - the core and the CAK. We found that the TFIIH core is initially located in the cytoplasm of syncytial blastoderm embryos, and that after mitotic division ten and until the cellular blastoderm stage, the core moves from the cytoplasm to the nucleus. By contrast, the CAK complex is mostly cytoplasmic during cellularization and during gastrulation. However, both components are positioned at promoters of genes that are activated at transcription onset. Later in development, the CAK complex becomes mostly nuclear and co-localizes in most chromosomal regions with the TFIIH core, but not in all sites, suggesting that the CAK complex could have a TFIIH-independent role in transcription of some loci. We also demonstrate that even though the CAK and the core coexist in the early embryo cytoplasm, they do not interact until they are in the nucleus and suggest that the complete assembly of the ten subunits of TFIIH occurs in the nucleus at the mid-blastula transition. In addition, we present evidence that suggests that DNA helicase subunits XPB and XPD are assembled in the core when they are transported into the nucleus and are required for the onset of transcription.