Cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-O-methyl derivative

The cytotoxicity of nine naphthoquinones (NQ) was assayed against HL-60 (leukaemia), MDA-MB-435 (melanoma), SF-295 (brain) and HCT-8 (colon), all human cancer cell lines, and peripheral blood mononuclear cells (PBMC), as representatives of normal cells, after 72 h of incubation. 5-Methoxy-1,4-naphthoquinone was the most active compound, showing IC₅₀ values in the range of 0.31 (1.7 μM) in HL-60 to 0.88 μg/mL (4.7 μM) in SF-295 and IC₅₀ of 0.69 μg/mL (3.7 μM) against PBMC. With the introduction of a bromo-substituent in position 2 or 3 of juglone, the IC₅₀ significantly decreased, regardless of the position on the NQ moiety. However, compared with juglone methyl ether, the halogen substitution decreased the activity. To further understand the mechanism underlying the cytotoxicity of 5-methoxy-1,4-naphthoquinone, studies involving DNA fragmentation, cell cycle analysis, phosphatidyl serine externalization, mitochondrial depolarization and activation of caspases 8 and 3/7 were performed in HL-60 cell line, using doxorubicin as a positive control. The results indicate that the cytotoxic 5-methoxy-1,4-naphthoquinone activates caspases 8 and 3/7 and thus induces apoptosis independent of mitochondria.     

A novel butyrylcholinesterase from serum of Leporinus macrocephalus, a Neotropical fish

We show here that serum of piaussu, a Neotropical characin fish, has the highest butyrylcholinesterase activity so far described for humans and fish. To clarify whether this cholinesterase could protect piaussu against anticholinesterase pesticides by scavenging organophosphates, we purified it 1700-fold, with a yield of 80%. Augmenting concentrations (from 0.01 to 20 mM) of butyrylthiocholine activated it. The pure enzyme was highly inhibited by chlorpyriphos-oxon (ki=10,434x10(6) M-1 min-1) and by the specific butyrylcholinesterase inhibitor, isoOMPA (ki=45.7x10(6) M-1 min-1). Electrophoresis of total serum and 2-D electrophoresis of the purified cholinesterase showed that some enzyme molecules could circulate in piaussu serum as heterogeneously glycosylated dimers. The enzyme's N-terminal sequence was similar to sequences found for butyrylcholinesterase from sera of other vertebrates. Altogether, our data present a novel butyrylcholinesterase with the potential of protecting a fish from poisoning by organophosphates.     

Revisiting Patterns of Tree Species Composition and their Driving Forces in the Atlantic Forests of Southeastern Brazil

The elucidation of phytogeographic patterns and their drivers in biodiversity hotspots is essential to the study of ecology and the conservation of these areas. In 2000, an important study by Oliveira‐Filho and Fontes led to changes in the paradigms that define our understanding of the Atlantic Forest (Brazil). Here, our aim was to revisit this study using a more comprehensive set of environmental predictors, an updated and much larger tree species dataset and checklist, and more refined data analyses. We performed exploratory and confirmatory analyses, including the modeling of the spatial components with Moran's Eigenvector Maps, using data from 483 sites in southeastern Brazil, which encompass a total of 3546 species and 33 geo‐climatic variables. We observed strong floristic similarities between rain‐ and seasonal forests and a species distribution continuum across the main gradients. The environmental and spatial variables were significantly correlated with floristic patterns, and we demonstrated that the tree flora of the seasonal forests should no longer be considered a simple subset of the rain forest flora. The findings of the original paper were not only confirmed but we also unveiled additional, important phytogeographic patterns. We also reinforced the main conclusion of the paper that the Atlantic Forest concept must encompass all of the forest types east of the dry corridor in South America, a designation of utmost importance for the conservation of this biodiversity hotspot.     

Characterization of invasive fish species in a river transposition region: evolutionary chromosome studies in the genus <Emphasis Type="Italic">Hoplias</Emphasis> (Characiformes,Erythrinidae)

Karyotypic analyses were performed in fishes from the genus Hoplias (H. malabaricus and H. lacerdae groups) from the São Francisco River basin (Brazil), in an impacted region by a river transposition which altered the local ecology and fish fauna. The karyotypes were investigated using chromosomal markers obtained from classic and molecular cytogenetics (Giemsa, CMA₃ and DAPI staining, C-banding, Ag-NORs, and FISH with 18S rDNA, 5S rDNA and 5SHindIII satellite DNA probes). Two karyotypic forms were found for the H. malabaricus group—karyomorph F, corresponding to the native form from the São Francisco River basin, and karyomorph A, corresponding to the invading form from the Upper Paraná River basin. Specimens from the H. lacerdae group exhibited striking chromosome differences in relation to the H. malabaricus group, thereby enabling good cytotaxonomic characterization and inferences regarding the karyotype evolution of these groups.     

The urea cycle in the liver of arthritic rats

The urea cycle in the liver of adjuvant-induced arthritic rats was investigated using the isolated perfused liver. Urea production in livers from arthritic rats was decreased during substrate-free perfusion and also in the presence of the following substrates: alanine, alanine + ornithine, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine but increased when arginine and citrulline + aspartate were the substrates. No differences were found with ammonia + aspartate, ammonia + aspartate + glutamate, aspartate, aspartate + glutamate and citrulline. Ammonia consumption was smaller in the arthritic condition when the substance was infused together with lactate or pyruvate but higher when the substance was simultaneously infused with aspartate or aspartate + glutamate. Glucose production tended to correlate with the smaller or higher rates of urea synthesis. Blood urea was higher in arthritic rats (+25.6%), but blood ammonia was lower (–32.2%). Critical for the synthesis of urea from various substrates in arthritic rats seems to be the availability of aspartate, whose production in the liver is probably limited by both the reduced gluconeogenesis and aminotransferase activities. This is indicated by urea synthesis which was never inferior in the arthritic condition when aspartate was exogenously supplied, being even higher when both aspartate and citrulline were simultaneously present. Possibly, the liver of arthritic rats has a different substrate supply of nitrogenous compounds. This could be in the form of different concentrations of aspartate or other aminoacids such as citrulline or arginine (from the kidneys) which allow higher rates of hepatic ureogenesis.     

Construction and Characterisation of a Genetically Engineered Escherichia coli Strain for the Epoxide Hydrolase-catalysed Kinetic Resolution of Epoxides

The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis, despite the formation of Eph1 inclusion bodies. Optimization of cultivation conditions and co-expression of molecular chaperones resulted in a further increase in activity and a reduction of the inclusion bodies formation, respectively. Compared to Rhodotorula glutinis cells and cell extracts, a total increase in Eph1 activity of over 200 times was found for both Escherichia coli cells and crude enzyme preparations of these cells. The improved conditions for recombinant Eph1 production were used to demonstrate the Eph1-catalysed kinetic resolution of a new Eph1 substrate, 1-oxaspiro[2.5]octane-2-carbonitrile.     

Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species

Active oxygen species are suspected as being a cause of the cellular damage that occurs at the site of inflammation. Phagocytic cells accumulate at these sites and produce superoxide ion, hydrogen peroxide and hydroxyl radical. The ultimate killing species, the cellular target and the mechanism whereby the lethal injury is produced are unknown. We exposed mouse fibroblasts to xanthine oxidase and acetaldehyde, a system which mimics the membrane of phagocytic cells in terms of production of oxygen species. We observed that the generation of these species produced DNA strand breaks and cellular death. The metal chelator o-phenanthroline completely abolished the former effect, and at the same time it effectively protected the cells from lethal injuries. Because complexing iron o-phenanthroline prevents the formation of hydroxyl radical by the Fendon reaction (Fe(II) + H2O2----Fe(III) + OH- + OH.), it is proposed that most of the cell death and DNA damage are brought about by OH radical, produced from other species by iron-mediated reactions.     

Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles

Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F₁-ATPase -subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and -glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial -presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immuno-detection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.     

Third molar agenesis in a trihybrid Brazilian population

The frequency of missing third molars is about eight per cent per quadrant in a sample of 490 males of the mixed White/Negro/Indian population of Natal, Brazil. Absence of the four third molars appears in two per cent of the individuals of the sample. The degree of asymmetry for the trait was not marked. No differences were observed when the persons were separated on morphological grounds as Whites or Negroids suggesting that these segregants may be genetically homogeneous in relation to this and other characteristics.