Effect of Oxidation on the Structure of Human Low- and High-Density Lipoproteins

This work presents a controlled study of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) structural changes due to in vitro oxidation with copper ions. The changes were studied by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) techniques in the case of LDL and by SAXS, DLS, and Z-scan (ZS) techniques in the case of HDL. SAXS data were analyzed with a to our knowledge new deconvolution method. This method provides the electron density profile of the samples directly from the intensity scattering of the monomers. Results show that LDL particles oxidized for 18 h show significant structural changes when compared to nonoxidized particles. Changes were observed in the electrical density profile, in size polydispersity, and in the degree of flexibility of the APO-B protein on the particle. HDL optical results obtained with the ZS technique showed a decrease of the amplitude of the nonlinear optical signal as a function of oxidation time. In contrast to LDL results reported in the literature, the HDL ZS signal does not lead to a complete loss of nonlinear optical signal after 18 h of copper oxidation. Also, the SAXS results did not indicate significant structural changes due to oxidation of HDL particles, and DLS results showed that a small number of oligomers formed in the sample oxidized for 18 h. All experimental results for the HDL samples indicate that this lipoprotein is more resistant to the oxidation process than are LDL particles.     

Volatile and molecular analysis of Juniperus brevifolia (Seub.) Antoine,an Azorean endemic species

Chemical and molecular analyses were performed for the characterization of Juniperus brevifolia (Seub.) Antoine (Cupressaceae), an Azorean endemic species. Forty-two individual twig samples were collected at seven Azorean islands (S. Miguel, Terceira, S. Jorge, Pico, Faial, Flores and Corvo). Volatile compounds were isolated by distillation–extraction and analyzed by GC and GC-MS. The volatile oils consisted mainly of monoterpene hydrocarbons (84–96%), limonene (33–87%), α-pinene (1–48%) and β-myrcene (1–5%) being the main components, and cluster analysis showed a high correlation among all samples (S_corr = 0.84). DNA fingerprinting was performed using thirty-seven RAPD and eleven ISSR primers, generating 881 polymorphic bands. Cluster analysis showed a moderate correlation among accessions (S_Dice = 0.43), which grouped according to their geographical origin. Chemical and molecular data did not show superimposable clustering profiles. Although the molecular approaches tested were useful in assessing genetic diversity, no straight correlation could be drawn between chemical and molecular data sets.     

The conserved and divergent roles of carbonic anhydrases in the filamentous fungi Aspergillus fumigatus and Aspergillus nidulans

Carbon dioxide (CO₂) and its hydration product bicarbonate (HCO₃^‐) are essential molecules in various physiological processes of all living organisms. The reversible interconversion between CO₂ and HCO₃^‐ is in equilibrium. This reaction is slow without catalyst, but can be rapidly facilitated by Zn²⁺‐metalloenzymes named carbonic anhydrases (CAs). To gain an insight into the function of multiple clades of fungal CA, we chose to investigate the filamentous fungi Aspergillus fumigatus and A. nidulans. We identified four and two CAs in A. fumigatus and A. nidulans, respectively, named cafA‐D and canA‐B. The cafA and cafB genes are constitutively, strongly expressed whereas cafC and cafD genes are weakly expressed but CO₂‐inducible. Heterologous expression of the A. fumigatus cafB, and A. nidulans canA and canB genes completely rescued the high CO₂‐requiring phenotype of a Saccharomyces cerevisiaeΔnce103 mutant. Only the ΔcafAΔcafB and ΔcanB deletion mutants were unable to grow at 0.033% CO₂, of which growth defects can be restored by high CO₂. Defects in the CAs can affect Aspergilli conidiation. Furthermore, A. fumigatusΔcafA, ΔcafB, ΔcafC, ΔcafD and ΔcafAΔcafB mutant strains are fully virulent in a low‐dose murine infection.     

Zinc and Copper Concentrations in Human Preterm Milk

Zinc and copper are important trace elements in the nutrition of preterm infants. This study determined and compared the concentrations of zinc and copper in preterm milk of mothers receiving and not receiving zinc supplementation diets. The effects of maternal supplementation on the blood levels and anthropometric parameters were evaluated. Thirty-eight mothers and their preterm infants were enrolled in the study. Eighteen mothers were given a daily supplementation of 50 mg zinc, whereas the other 20 were not. Zinc and copper levels in milk were determined at 15-day intervals, as were blood zinc levels along with anthropometric parameters. Zinc and copper contents were determined by atomic absorption spectrophotometry. No significant differences were found between groups either for zinc values in maternal milk and infant blood or for anthropometric measurements. A pronounced decrease in copper levels was observed in mothers receiving supplemental zinc. Zinc supplementation given to mothers who were breastfeeding preterm infants had no significant effect on zinc secretion in milk.     

Presence of Cryptosporidium spp. and Giardia duodenalis in Drinking Water Samples in the North of Portugal

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and β,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.     

Characterization of heme as activator of Toll-like receptor 4

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-alpha secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-alpha and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.     

Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro

The aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM - control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.     

Reproductive biology of the lane snapper,Lutjanus synagris (Linnaeus 1758) (Perciformes,Lutjanidae), in the Maranhão continental shelf,Northeast of Brazil

Environmental Biology of Fishes - To clarify the reproductive activity of lane snapper, Lutjanus synagris, a total of 359 specimens of lane snapper were collected in partnership with an artisanal...