Mitochondrial respiratory chain dysfunction, a non-receptor-mediated effect of synthetic PPAR-ligands: biochemical and pharmacological implications

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation, and cell differentiation. Many of their biological activities have been studied by using selective synthetic activators (mainly fibrates and thiazolidinediones) which have been already employed in therapeutic protocols. Both kinds of drugs, however, showed pharmacotoxicological profiles, which cannot be ascribed by any means to receptor activation. To better understand these non-receptorial or extrareceptorial aspects, the effect of different PPAR-ligands on the metabolic status of human HL-60 cell line has been investigated. At this regard, NMR analysis of cell culture supernatants was accomplished in order to monitor modifications at the level of cell metabolism. Cell growth and chemiluminescence assays were employed to verify cell differentiation. Results showed that all the considered PPAR-ligands, although with different potencies and independently from their PPAR binding specificity, induced a significant derangement of the mitochondrial respiratory chain consisting in a strong inhibition of NADH-cytochrome c reductase activity. This derangement has been shown to be strictly correlated to the adaptive metabolic modifications, as evidenced by the increased formation of lactate and acetate, due to the stimulation of anaerobic glycolysis and fatty acid beta-oxidation. It is worthy noting that the mitochondrial dysfunction appeared also linked to the capacity of any given PPAR-ligand to induce cell differentiation. These data could afford an explanation of biochemical and toxicological aspects related to the therapeutic use of synthetic PPAR-ligands and suggest a revision of PPAR pathophysiologic mechanisms.     

Effects of food limitation and temperature on cladocerans from a tropical Brazilian lake

Food limitation was tested in the laboratory by individual growth and reproduction of two cladoceran species, Ceriodaphnia richardi and Daphnia gessneri, from the shallow tropical Brazilian Lake Monte Alegre. The cladocerans were fed cultivated green alga Scenedesmus spinosus in concentrations of 0.20, 0.10, 0.05, and 0.025 mg C l⁻¹. Higher biomass and growth rates occurred in the two highest-food concentrations; the two lowest ones negatively affected clutch size and first reproduction. The threshold food concentration is lower than 0.025 mg C l⁻¹ and the incipient limiting level is a value between 0.10 and 0.20 mg C l⁻¹. The largest species, D. gessneri, was more sensitive to low food concentrations. The effects of low and high temperatures (19 and 27°C) were evaluated by life table experiments with three cladocerans from the lake—Daphnia ambigua, D. gessneri, and Moina micrura—with no food limitation (1 mg C l⁻¹ of S. spinosus). Higher population growth rates for the three species were found at 27°C; better performance in most life table parameters was observed for the former two species at the highest temperature, D. gessneri being the most sensitive to the lowest temperature. There are indications that temperature is an important abiotic factor that constrains populations of cladocerans for a short period in winter in the lake, when temperature decreases to 18–19°C. However, its influence cannot be separated from a biotic factor such as food, whose effect is stronger in the cool season, when concentrations are lower and contribution of inedible algae is relatively higher.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.     

Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline

Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.     

Recovery of Flavobacterium psychrophilum viable cells using a charcoal-based solid medium

AIMS: Flavobacterium psychrophilum is the etiological agent of the cold-water disease in salmonids. This micro-organism is somewhat fastidious being difficult to isolate and culture. The aim of this study was to develop a new solid medium which improves the recovery of viable cells from a sample. METHODS AND RESULTS: Six different media [nutrient agar (NA), NA + charcoal (NAC), enriched Anacker Ordal serum (EAOS), EAOS supplemented with aromatic compounds (EAOSa), EAOS with activated charcoal (EAOC) and EAOC supplemented with aromatic compounds (EAOCa)], three of them containing activated charcoal, were used to recover isolated colonies from a diluted sample of Fl. psychrophilum THC02-90. Pair wise comparisons between different media were carried out using the test of bootstrap to determine the best solid medium and if the presence of activated charcoal increased the number of colonies. The results showed that activated charcoal improved the recovery of viable cells in all the cases and NAC was slightly better than EAOCa but more variable. CONCLUSIONS: Activated charcoal has a great capacity of absorption of toxic compounds and it has no nutritional value, so the problems to culture and isolate Fl. psychrophilum are in part due to an inhibition phenomenon. The use of EAOCa can overcome some of these problems. SIGNIFICANCE AND IMPACT OF THE STUDY: The improvement in Fl. psychrophilum cultivation will facilitate physiological, biochemical and genetic studies with this bacterium.     

Supramolecular structure of self-assembling alamethicin analog studied by ESR and PELDOR

Three analogs of alamethicin F50/5, labelled with the TOAC (='2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid') spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron-Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, alamethicin forms homogeneous solutions. In the weakly polar chloroform/toluene 1 : 1 mixture, however, this peptide forms aggregates that are detectable at 293 K by ESR in liquid solution, as well as by PELDOR in frozen, glassy solution at 77 K. In liquid solution, free alamethicin molecules and their aggregates show rotational-mobility correlation times tau(r) of 0.87 and 5.9 ns, resp. Based on these values and analysis of dipole-dipole interactions of the TOAC labels in the aggregates, as determined by PELDOR, the average number N of alamethicin molecules in the aggregates is estimated to be less than nine. A distance-distribution function between spin labels in the supramolecular aggregate was obtained. This function exhibits two maxima: a broad one at a distance of 3.0 nm, and a wide one at a distance of ca. 7 nm. A molecular-dynamics (MD)-based model of the aggregate, consisting of two parallel tetramers, each composed of four molecules arranged in a 'head-to-tail' fashion, is proposed, accounting for the observed distances and their distribution.     

Evaluation of Antifungal Activity of Medicinal Plant Extracts Against Oral <Emphasis Type="Italic">Candida albicans</Emphasis> and Proteinases

Proteinases produced by Candida albicans are one kind of virulence factor expressed that contribute to adherence and invasion of host tissue. Proteinase inhibitor of human immunodeficiency virus in experimental candidiasis suggested reduction in fungal infection, and medicinal plants could be a source of alternative agent to prevent diseases. In this study, we investigated the production of proteinases by C. albicans from clinical isolates and the action of plant extracts against strains of C. albicans and its synthesized proteinases, comparing with antifungal fluconazole and amphotericin B and proteinase inhibitors pepstatin A, amprenavir, and ritonavir. The results reported here showed that these extracts have a certain kind of action and that the search for new antifungal agents could be found at the plants.