Purification and properties of the enzyme 6-phosphogluconate dehydrogenase from Dicentrarchus labrax L. liver

The enzyme 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP+ oxidoreductase, decarboxylating EC 1.1.1.44) from bass liver has been purified to over 95% of homogeneity by gel filtration, affinity and ion exchange chromatographies. The apparent molecular weight was estimated by gel filtration chromatography to about 100,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed to be a dimeric protein. The effect of pH and kinetic properties were studied.     

The Expanded mtDNA Phylogeny of the Franco-Cantabrian Region Upholds the Pre-Neolithic Genetic Substrate of Basques

The European genetic landscape has been shaped by several human migrations occurred since Paleolithic times. The accumulation of archaeological records and the concordance of different lines of genetic evidence during the last two decades have triggered an interesting debate concerning the role of ancient settlers from the Franco-Cantabrian region in the postglacial resettlement of Europe. Among the Franco-Cantabrian populations, Basques are regarded as one of the oldest and more intriguing human groups of Europe. Recent data on complete mitochondrial DNA genomes focused on macrohaplogroup R0 revealed that Basques harbor some autochthonous lineages, suggesting a genetic continuity since pre-Neolithic times. However, excluding haplogroup H, the most representative lineage of macrohaplogroup R0, the majority of maternal lineages of this area remains virtually unexplored, so that further refinement of the mtDNA phylogeny based on analyses at the highest level of resolution is crucial for a better understanding of the European prehistory. We thus explored the maternal ancestry of 548 autochthonous individuals from various Franco-Cantabrian populations and sequenced 76 mitogenomes of the most representative lineages. Interestingly, we identified three mtDNA haplogroups, U5b1f, J1c5c1 and V22, that proved to be representative of Franco-Cantabria, notably of the Basque population. The seclusion and diversity of these female genetic lineages support a local origin in the Franco-Cantabrian area during the Mesolithic of southwestern Europe, ∼10,000 years before present (YBP), with signals of expansions at ∼3,500 YBP. These findings provide robust evidence of a partial genetic continuity between contemporary autochthonous populations from the Franco-Cantabrian region, specifically the Basques, and Paleolithic/Mesolithic hunter-gatherer groups. Furthermore, our results raise the current proportion (≈15%) of the Franco-Cantabrian maternal gene pool with a putative pre-Neolithic origin to ≈35%, further supporting the notion of a predominant Paleolithic genetic substrate in extant European populations.     

Genome-wide analysis of the SET DOMAIN GROUP family in grapevine

The SET DOMAIN GROUP (SDG) proteins represent an evolutionarily-conserved family of epigenetic regulators present in eukaryotes and are putative candidates for the catalysis of lysine methylation in histones. Plant genomes analyses of this family have been performed in arabidopsis, maize, and rice and functional studies have shown that SDG genes are involved in the control of plant development. In this work, we describe the identification and structural characterization of SDG genes in the Vitis vinifera genome. This analysis revealed the presence of 33 putative SDG genes that can be grouped into different classes, as it has been previously described for plants. In addition to the SET domain, the proteins identified possessed other domains in the different classes. As part of our study regarding the growth and development of grapevine, we selected eight genes and their expression levels were analyzed in representative vegetative and reproductive organs of this species. The selected genes showed different patterns of expression during inflorescence and fruit development, suggesting that they participate in these processes. Furthermore, we showed that the expression of selected SDGs changes during viral infection, using as a model Grapevine Leafroll Associated Virus 3-infected symptomatic grapevine leaves and fruits. Our results suggest that developmental changes caused by this virus could be the result of alterations in SDG expression.     

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

Oxidative metabolism of cholesterol precursors: sensitivity to ketoconazole, an inhibitor of cytochrome P-450

The effects of ketoconazole, an inhibitor of cytochrome P-450, on the metabolism of the cholesterol precursors lanosterol, dihydrolanosterol, lanost-8-en-3 beta,32-diol, and 3 beta-hydroxylanost-8-en-32-al were investigated in subcellular fractions of rat liver and in rat hepatocytes in culture. At low (1-2 microM) concentrations of the drug, the oxidative demethylation of lanosterol was inhibited by about 70% in the subcellular fractions but there was no effect on the metabolism of the 3 beta, 32-diol or the 32-aldehyde. Higher drug concentrations (10-20 microM) were required to inhibit the oxidative metabolism of these cholesterol precursors. Similar results were obtained during longer-term incubations using hepatocytes in culture medium, but higher concentrations of ketoconazole were required to effect the same degree of inhibition of each precursor. In the subcellular fractions, dihydrolanosterol, the 3 beta,32-diol and the 32-aldehyde were each metabolized to more polar sterols, in addition to cholesterol. Ketoconazole also inhibited the formation of these polar substances.     

The use of functional analysis of the ribosome as a tool to determine archaebacterial phylogeny

Forty different antibiotics with diverse kingdom and functional specificities were used to measure the functional characteristics of the archaebacterial translation apparatus. The resulting inhibitory curves, which are characteristic of the cell-free system analyzed, were transformed into quantitative values that were used to cluster the different archaebacteria analyzed. This cluster resembles the phylogenetic tree generated by 16S rRNA sequence comparisons. These results strongly suggest that functional analysis of an appropriate evolutionary clock, such as the ribosome, is of intrinsic phylogenetic value. More importantly, they indicate that the study of the nexus between genotypic and phenotypic (functional) information may shed considerable light on the evolution of the protein synthetic machinery.