Loading...
6 results
Search Results
Now showing 1 - 6 of 6
- DNA cleavage activity of VIVO(acac)2 and derivativesPublication . Butenko, Nataliya; Tomaz, Ana Isabel; Nouri, Ofelia; Escribano, Esther; Moreno, Virtudes; Gama, Sofia; Ribeiro, Vera; Telo, João Paulo; Pessoa, João Costa; Cavaco, Isabel Maria Palma AntunesThe DNA cleavage activity of several b-diketonate vanadyl complexes is examined. Vanadyl acetylacetonate,VIVO(acac)2, 1, shows a remarkable activity in degrading plasmid DNA in the absence of any activating agents, air and photoirradiation. The cleaving activity of several related complexes VIVO(hd)2(2, Hhd = 3,5-heptanedione), VIVO(acac-NH2)2 (3, Hacac-NH2 = acetoacetamide) and VIVO(acac-NMe2)2(4, Hacac-NMe2 = N,N-dimethylacetoacetamide) is also evaluated. It is shown that 2 exhibits an activity similar to 1, while 3 and 4 are much less efficient cleaving agents. The different activity of the complexes is related to their stability towards hydrolysis in aqueous solution, which follows the order 1 2 3 4.The nature of the pH buffer was also found to be determinant in the nuclease activity of 1 and 2. In a phosphate buffered medium DNA cleavage by these agents is much more efficient than in tris, hepes,mes or mops buffers. The reaction seems to take place through a mixed mechanism, involving the formation of reactive oxygen species (ROS), namely OH radicals, and possibly also direct cleavage at phosphodiester linkages induced by the vanadium complexes.
- Electrochemical DNA sensor for detection of single nucleotide polymorphismsPublication . Marques, L. P. J.; Cavaco, Isabel Maria Palma Antunes; Pinheiro, J. P.; Ribeiro, Vera; Ferreira, GuilhermeIn recent years there has been an increased interest in using biosensors for the recognition and monitoring of molecule interactions. DNA sensors and gene chips are particularly relevant for directly applying the information gathered from the genome projects. In this work electrochemical techniques are used to develop methodologies to detect DNA polymorphisms in human genes using cytochrome P450 3A4 (CYP3A4) as a model gene. CYP3A4*1B oligonucleotides were immobilized on the surface of a gold electrode and hybridized with fully complementary oligonucleotide sequences as well as with mismatched sequences corresponding to the CYP3A4*1A reference sequence. The methodology developed is based on double-stranded DNA’s ability to transport charge along nucleotide stacking. The perturbation of the double helix pi-stack introduced by a mismatched nucleotide reduces electron flow and can be detected by measuring the attenuation of the charge transfer. The methodology developed could identify CYP3A4*1A homozygotes by the 5 μC charge attenuation observed when compared with DNA samples containing at least one CYP3A4*1B allele.
- Bioenergetic cues shift FXR splicing towards FXR alpha 2 to modulate hepatic lipolysis and fatty acid metabolismPublication . Correia, Jorge; Massart, Julie; de Boer, Jan Freark; Porsmyr-Palmertz, Margareta; Martinez-Redondo, Vicente; Agudelo, Leandro Z.; Sinha, Indranil; Meierhofer, David; Ribeiro, Vera; Bjornholm, Marie; Sauer, Sascha; Dahlman-Wright, Karin; Zierath, Juleen R.; Groen, Albert K.; Ruas, Jorge L.Objective: Farnesoid X receptor (FXR) plays a prominent role in hepatic lipid metabolism. The FXR gene encodes four proteins with structural differences suggestive of discrete biological functions about which little is known. Methods: We expressed each FXR variant in primary hepatocytes and evaluated global gene expression, lipid profile, and metabolic fluxes. Gene delivery of FXR variants to Fxr(-/-) mouse liver was performed to evaluate their role in vivo. The effects of fasting and physical exercise on hepatic Fxr splicing were determined. Results: We show that FXR splice isoforms regulate largely different gene sets and have specific effects on hepatic metabolism. FXR alpha 2 (but not alpha 1) activates a broad transcriptional program in hepatocytes conducive to lipolysis, fatty acid oxidation, and ketogenesis. Consequently, FXR alpha 2 decreases cellular lipid accumulation and improves cellular insulin signaling to AKT. FXR alpha 2 expression in Fxr(-/-) mouse liver activates a similar gene program and robustly decreases hepatic triglyceride levels. On the other hand, FXRa1 reduces hepatic triglyceride content to a lesser extent and does so through regulation of lipogenic gene expression. Bioenergetic cues, such as fasting and exercise, dynamically regulate Fxr splicing in mouse liver to increase Fxr alpha 2 expression. Conclusions: Our results show that the main FXR variants in human liver (alpha 1 and alpha 2) reduce hepatic lipid accumulation through distinct mechanisms and to different degrees. Taking this novel mechanism into account could greatly improve the pharmacological targeting and therapeutic efficacy of FXR agonists. (C) 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).
- Plasmodium infection alters Anopheles gambiae detoxification gene expressionPublication . Felix, Rute C.; Mueller, Pie; Ribeiro, Vera; Ranson, Hilary; Silveira, HenriqueBackground: Anopheles gambiae has been shown to change its global gene expression patterns upon Plasmodium infection. While many alterations are directly related to the mosquito's innate immune response, parasite invasion is also expected to generate toxic by-products such as free radicals. The current study aimed at identifying which loci coding for detoxification enzymes are differentially expressed as a function of Plasmodium berghei infection in midgut and fat body tissues. Results: Using a custom-made DNA microarray, transcript levels of 254 loci primarily belonging to three major detoxification enzyme families (glutathione S-transferases, cytochrome P450 monooxygenases and esterases) were compared in infected and uninfected mosquitoes both during ookinete invasion and the release of sporozoites into the hemocoel. The greatest changes in gene expression were observed in the midgut in response to ookinete invasion. Interestingly, many detoxification genes including a large number of P450s were down-regulated at this stage. In the fat body, while less dramatic, gene expression alterations were also observed and occurred during the ookinete invasion and during the release of sporozoites into the hemocoel. While most gene expression changes were tissue-related, CYP6M2, a CYP previously associated with insecticide resistance, was over-expressed both in the midgut and fat body during ookinete invasion. Conclusions: Most toxicity-related reactions occur in the midgut shortly after the ingestion of an infected blood meal. Strong up-regulation of CYP6M2 in the midgut and the fat body as well as its previous association with insecticide resistance shows its broad role in metabolic detoxification.
- Pharmacogenetic analysis of inter-ethnic variability in the uptake transporter SLCO1B1 gene in Colombian, Mozambican, and Portuguese populationsPublication . Nega, Mulata Haile; Berhe, Derbew F.; Ribeiro, VeraBackground Statin-induced myopathy is reported to be associated with the solute carrier organic anion transporter family member 1B1 gene single nucleotide polymorphism, c.521 T > C. There is no epidemiologic data on this gene polymorphism in several countries. Therefore, this study aimed at assessing the genotype and allele frequencies of the gene variant in three countries. Methods This study involved healthy individuals from Colombia, Mozambique, and Portugal. Genomic DNA was isolated from blood samples using the Qiamp DNA Extraction Kit (Qiagen). The isolated DNA was genotyped using novel Polymerase Chain Reaction—Restriction Fragment Length Polymorphism. Microstat and GraphPad QuickCal software were used for the Chi-square test and the evaluation of Hardy–Weinberg equilibrium respectively. Results A total of 181 individuals’ blood samples were analyzed. Overall, the TT (74.0%) genotype was the highest and the CC (7.8%) was the lowest. Country wise genotypic frequencies were Colombia 47(70.2%) TT, 12(17.9%) TC and 8(11.9%) CC; Mozambique 47(88.7%) TT, 5(9.4%) TC, and 1(1.9%) CC; and Portugal 40(65.6%) TT, 16(26.2%) TC, and 5(8.2%) CC. The reference (T) allele was highest among Mozambicans (93.4%) compared to Colombians (79.1%) and Portuguese (78.7%). Mozambicans showed statistically significant genotypic and allelic frequency differences compared to Colombians (p < 0.01) and Portuguese (p < 0.01). Conclusions Overall and country-wise, CC genotype was less frequent and it is relatively high for Colombians and Portuguese populations. This finding may imply statins risk–benefit variability associated with CC genotype among these populations that needs further understanding.
- Cucurbiturils as supramolecular inhibitors of DNA restriction by type II endonucleasesPublication . Parente Carvalho, Catia; Norouzy, Amir; Ribeiro, Vera; Nau, Werner M.; Pischel, UweCucurbiturils (CB6 and CB7) were shown to inhibit the enzymatically catalyzed restriction of plasmids and linear DNA. This effect can be inverted by supramolecular masking of the macrocycles through competitive complexation with polyamines. These experiments provide supramolecular control of biocatalytic processes.