Vitamin C modulation of H2O2-induced damage and iron homeostasis in human cells

Vitamin C (ascorbic acid, AA) is an important antioxidant in human plasma. It is clear, however, that AA has other important, nonantioxidant roles in cells. Of particular interest is its involvement in iron metabolism, since AA enhances dietary iron absorption, increases the activity of Fe(2+)-dependent cellular enzymes, promotes Fenton reactions in vitro, and was reported to have deleterious effects in individuals with iron overload. Nevertheless, the ability of AA to modulate iron metabolism and enhance iron-dependent damage in cells, tissues, and organisms has not been fully elucidated. Here we investigated the effect of AA on iron-mediated oxidative stress in normal human fibroblasts. Incubation with physiologically relevant concentrations of AA was not harmful but sensitised cells toward H(2)O(2)-induced, iron-dependent DNA strand breakage and cell death. We also report that AA increased the levels of intracellular catalytic iron and concomitantly modulated the expression of two well-established iron-regulated genes, ferritin and transferrin receptor. In summary, we present evidence of a novel, nonantioxidant role of AA in human cells, where it increases iron availability and enhances ROS-mediated, iron-dependent damage. We suggest that AA may exacerbate the deleterious effects of metals in vivo and promote normal tissue injury in situations associated with elevated ROS production.     

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O₂ ^•− source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O₂ ^•− molecule and half a H₂O₂ molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min⁻¹, respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 μM⁻¹ min⁻¹) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 μM⁻¹ min⁻¹). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O₂ ^•− source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O₂ ^•− than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.     

Genetic diversity of three endangered species of <Emphasis Type="Italic">Echium</Emphasis> L. (Boraginaceae) endemic to Cape Verde Islands

Echium hypertropicum, E. stenosiphon and E. vulcanorum are the three endemics representative of the genus Echium (Boraginaceae) in Cape Verde archipelago. The aim of this study is to provide a first attempt at estimating genetic diversity among natural populations of these endangered Echium species based on RAPD, so as to provide data available for future appropriate strategies for their conservation. PCO and UPGMA of RAPD analysis suggests a close genetic proximity between the Southern endemic species (E. hypertropicum and E. vulcanorum) and shows that the levels of polymorphism strongly differ between these two Echium species (27 and 29% respectively) and E. stenosiphon (74%), the Northern endemic species. Mantel test also corroborates a close genetic proximity between genetic and geographic data. Population genetic analysis of E. stenosiphon revealed low levels of gene flow between islands (Nm = 0.32) being S. Nicolau the most isolated as evident in PCO. Furthermore the differentiation between groups of individuals belonging to putative subspecies was tested by AMOVA. According to our results there is no genetic basis to consider the two subspecies of E. stenosiphon namely ssp. stenosiphon and ssp. lindbergii. Our results enable us to suggest that E. stenosiphon must be ranked as a Threatened species. Measures aiming at conservation of E. hypertropicum and E. vulcanorum must be implemented at short-term taking into account the small number of existing plants and its low genetic variability.     

Apoptosis induction and modulation of P-glycoprotein mediated multidrug resistance by new macrocyclic lathyrane-type diterpenoids

The macrocyclic lathyrane diterpenes, latilagascenes D-F (1-3) and jolkinol B (4), were isolated from the methanol extract of Euphorbia lagascae, and evaluated for multidrug resistance reversing activity on mouse lymphoma cells. All compounds displayed very strong activity compared with that of the positive control, verapamil. The structure-activity relationship is discussed. The evaluation of compounds 1 and 4, and of latigascenes A-C (5-7), isolated from the same species, as apoptosis-inducers was also carried out. Compound 1 was the most active. Furthermore, in the model of combination chemotherapy, the interaction between the doxorubicine and latilagascene B (6) was studied in vitro, on human MDR1 gene transfected mouse lymphoma cells, showing that the type of interaction was synergistic. Latilagascenes D-F (1-3) are new compounds whose structures were established on the basis of spectroscopic methods, including 2D NMR experiments (COSY, HMQC, HMBC and NOESY).     

Synthesis of neutral and cationic tripyridylporphyrin-D-galactose conjugates and the photoinactivation of HSV-1

Neutral and cationic tripyridylporphyrin-D-galactose conjugates were synthesized and their antiviral activity against herpes simplex virus type 1 (HSV-1) was evaluated. At non-cytotoxic concentrations the studied compounds show significant antiviral activity after photoactivation. The influence of photoactivation on drug treated cells was also analyzed, at different times of infection with HSV-1, for a neutral (1b) and a cationic glycoporphyrin (3b) derivative. The results show that the inhibition of the viral yield is more dependent on photoactivation for compound 1b than for compound 3b. These two compounds also differ in the inhibitory effect during the viral replicative cycle: while compound 3b inhibits the viral yield at all the addition times assayed, compound 1b is more efficient in later times of infection.     

PKC anchoring to GluR4 AMPA receptor subunit modulates PKC-driven receptor phosphorylation and surface expression

Changes in the synaptic content of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors lead to synaptic efficacy modifications, involved in synaptic plasticity mechanisms believed to underlie learning and memory formation. Early in development, GluR4 is highly expressed in the hippocampus, and GluR4-containing AMPA receptors are inserted into synapses. During synapse maturation, the number of AMPA receptors at the synapse is dynamically regulated, and both addition and removal of receptors from postsynaptic sites occur through regulated mechanisms. GluR4 delivery to synapses in rat hippocampal slices was shown to require protein kinase A (PKA)-mediated phosphorylation of GluR4 at serine 842 (Ser842). Protein kinase C (PKC) can also phosphorylate Ser842, and we have shown that PKCgamma can associate with GluR4. Here we show that activation of PKC in retina neurons, or in human embryonic kidney 293 cells cotransfected with GluR4 and PKCgamma, increases GluR4 surface expression and Ser842 phosphorylation. Moreover, mutation of amino acids R821A, K825A and R826A at the GluR4 C-terminal, within the interacting region of GluR4 with PKCgamma, abolishes the interaction between PKCgamma and GluR4 and prevents the stimulatory effect of PKCgamma on GluR4 Ser842 phosphorylation and surface expression. These data argue for a role of anchored PKCgamma in Ser842 phosphorylation and targeting to the plasma membrane. The triple GluR4 mutant is, however, phosphorylated by PKA, and it is targeted to the synapse in CA1 hippocampal neurons in organotypic rat hippocampal slices. The present findings show that the interaction between PKCgamma and GluR4 is specifically required to assure PKC-driven phosphorylation and surface membrane expression of GluR4.     

High Metabolic Rates in Beach Cast Communities

Metabolic hotspots at land–water interfaces are important in supporting biogeochemical processes. Here we confirm the generality of land–aquatic interfaces as biogeochemical hot spots by extending this concept to marine beach cast materials. In situ atmospheric pCO_2, from a respiration chamber (10 cm in diameter and 20 cm high) inserted into wrack deposits, was determined using a high-precision (±1 ppm) non-dispersive infrared gas analyzer (EGM-4, PP-systems) at 1 minute recording intervals. The wrack deposits supported high metabolic activities, with CO₂ fluxes averaging (±SE) 6.62 ± 0.88 μmol C m⁻² s⁻¹, compared to median value of 0.98 μmol C m⁻² s⁻¹ (mean 2.21 ± 1.25 μmol C m⁻² s⁻¹) for bare sand adjacent to deposits. Wrack metabolic rates ranged 40-fold across beaches, from a minimum of 0.57 ± 0.22 μmol C m⁻² s⁻¹ to a maximum of 20.8 ± 5.04 μmol C m⁻² s⁻¹, both derived from beaches with deposits dominated by Sargassum. Rates tended to increase significantly (F test, P < 0.05) from the shoreline to reach maximum rates at about 10 m from the shoreline, declining sharply further from the shoreline, and increased with increasing thickness of the deposits (maximum about 10 cm deep), declining for thicker deposits. Wrack differing in composition had similar metabolic rates, although deposits consisting of a mixture of seagrass and algae tended to show somewhat higher rates. Our results show a meter square of wrack deposit supports a metabolic rate equivalent to that supported by 3 m² of living seagrass or macroalgal habitat. In wrack, the marine environment provides organic material and moisture and the land environment provides oxygen to render wrack ecosystems an efficient metabolic reactor. Intense wrack metabolism should also be conducive to organismal growth by supporting the development of a cryptic, but diverse wrack-based food web.     

Aspergilli and lignocellulosics: Enzymology and biotechnological applications

Abstract: Aspergilli are versatile ascomycetes that are able to transform at a rapid rate a wide spectrum of lignin-related aromatic compounds. While it is clear that these fungi can degrade phenolic and polysaccharide components from lignocellulosic material, the status regarding degradation of high-molecular mass lignins is controversial. This review compiles data from the literature as well as that from the authors' laboratory with the aim of clarifying this point. The main body of evidence points towards the inability of aspergilli alone to degrade lignin free of low-molecular mass contaminants. Nevertheless, the ability of this genus to efficiently degrade hemicelluloses makes it an essential participant in the complex microbial system necessary for wood decay under natural conditions. Aspergilli are known to overproduce high levels of hemicellulolytic enzymes. Out of the large array of these enzymes that act in concert to degrade lignocellulosic material, only endoxylanases of aspergilli are described in so far as these are the main activities required for enzyme-aided bleaching. The biochemical features of the endoxylanases from Aspergillus niger are briefly described as these serve to illustrate how a complex family of isozymes is necessary to deal with the structural and chemical heterogeneity of xylans. Emphasis is placed on the biotechnological applications of lignocellulosic materials transformed by aspergilli. The key application areas are biopulping and biobleaching where a reduction in the use of environmentally harmful chemicals traditionally used in the pulp and paper industry is envisaged. Waste water treatment represents another vast application area where aspergilli have been shown to be effective not only in colour removal but also in the bioconversion of potentially noxious substances into useful bioproducts.     

Filarids (Spirurida: Onchocercidae) in wild carnivores and domestic dogs from the Brazilian Atlantic forest

Onchocercidae nematodes are heteroxenous parasites with worldwide distribution, and some of the species associated to animals may present zoonotic potential. Climatic changes and anthropic influences on the environment may result in vectors’ proliferation, facilitating the spillover to humans and/or non-typical animal hosts. The Iguaçu National Park (PARNA Iguaçu), one of the most important Brazilian natural remanescents of Atlantic rainforest, is strongly affected by human activities such as tourism and agriculture. The complexity of this area is especially characterized by the close nexus between the rich wildlife, humans, and domestic animals, especially domestic dogs. Based on this, this research aimed to diagnose the Onchocercidae nematodes in wild carnivores and domestic dogs in the PARNA Iguaçu and the surrounding areas. For this, we collected 162 samples of seven species of wild carnivores and 225 samples of domestic dogs. The presence of microfilariae in the blood samples was diagnosed by the modified Knott’s test and molecular screening, and the specific identification was based on sequencing of the myoHC and hsp70 genes. Microfilariae were detected only in ring-tailed coatis, in which we found five species: Mansonella sp. 1, Mansonela sp. 2, Onchocercidade gen. sp. 1, Onchocercidade gen. sp. 2, and Dirofilaria immitis. The morphological analysis supported the molecular findings. The domestic dogs were parasitized by Acanthocheilonema reconditum, representing a new locality record for this species. Phylogenetic analysis showed high genetic similarity among the four undetermined species and Mansonella spp., Brugia spp., and Wuchereria bancrofti. The presence of D. immitis in ring-tailed coatis may be result of spillover from dogs, even though the parasite was not diagnosed in the sampled dogs. The presence of several undetermined Onchocercidae species indicates the necessity of continuous investigations on wild and domestic animals from Neotropical area, especially considering the growing anthropic influence on forest remnants.