Mechanism of action of levonorgestrel: in vitro metabolism and specific interactions with steroid receptors in target organs.

Levonorgestrel (LNG) is a synthetic steroid that displays potent progestional and androgenic effects but it lacks estrogen-like activity. To examine the mode of action of this progestin, we studied its metabolism in vitro in target organs and the specific interactions of LNG and its metabolites with putative steroid receptors. The results demonstrated that [³H]LNG was efficiently converted to A-ring reduced derivatives when incubated with rat hypothalamus and pituitary. Under optimal incubation conditions, [³H]5-dihydro LNG (5-LNG) and [³H]3-5-tetrahydro LNG (3,5-LNG) were identified as the major metabolic conversion products, while [³H]3ß, 5-LNG formation occured to a lesser extent. A-ring reduction of LNG was NADPH-dependent. Assessment of the relative binding affinities of LNG and its derivatives to progesterone (PR), androgen (AR) and estrogen (ER) receptors by displacement analysis revealed that unchanged LNG binds with high affinity to PR and AR but not to ER. 5-LNG exhibited a diminished though significant interactions with PR and an enhanced binding affinity for AR as compared with LNG, indicating that 5-reduction of LNG increases its affinity for AR. The most striking finding was that further reduction of the 5-LNG molecule at C-3 abolished its binding activity to PR, AR, and even to ER. The overall data provides a plausible explanation for the lack of estrogen agonistic action of LNG and for its potent progestational and androgenic effects.     

Effects of t-butyl-4-hydroxyanisole and other phenolic antioxidants on tumoral cells and Trypanosoma parasites

The antioxidant food additives 2(3)-tert-butyl-4-hydroxyanisole (BHA), 2,6-di(tert-butyl)-p-cresol (BHT) and the methyl and propyl esters of gallic acid inhibited Trypanosoma cruzi culture growth and oxygen consumption. The I50 values for growth and oxygen uptake with BHA were 0.284 and 0.400 and for BHT 0.083 and 0.235 mM, respectively. Moreover, BHA inhibited the respiration of several tumor cells, as well as of the procyclic and bloodstream trypomastigote forms of T. brucei brucei, with I50 in the range 0.29-0.52 mM. Inhibition of the parasites' oxygen uptake by BHA was not of the pure Michaelis-Menten type, but may be of a mixed form. It is postulated that these compounds are inhibitors because they resemble ubiquinone.     

In vitro and in vivo studies of Trypanosoma cruzi DNA polymerase

One major DNA polymerase has been purified and characterized from Trypanosoma cruzi. The enzyme has a sedimentation coefficient of 6.8 S corresponding to an approximate molecular weight of 180,000 assuming a globular shape. The enzyme recognizes activated DNA very efficiently, as well as synthetic polydeoxynucleotides, whereas poly rA-dT12 is very poorly utilized. Trypanosoma cruzi DNA polymerase is not inhibited at all by aphidicolin, while araCTP inhibits the enzyme very slightly. The purified enzyme is strongly inhibited by N-ethyl maleimide, dideoxyTTP, ethidium bromide and berenil. All our attempts to find a DNA polymerase sensitive to aphidicolin in vitro have failed, nor have we been able to find a low molecular weight DNA polymerase in this organism. However, when DNA synthesis was studied in whole trypanosomes, aphidicolin was shown to inhibit DNA synthesis more efficiently than ethidium bromide and berenil.     

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.     

Soil nutrient dissimilarity and litter nutrient limitation as major drivers of home field advantage in riparian tropical forests

Decomposition is a key process driving carbon and nutrient cycling in ecosystems worldwide. The home field advantage effect (HFA) has been found to accelerate decomposition rates when litter originates from “home” when compared to other (“away”) sites. It is still poorly known how HFA plays out in tropical, riparian forests, particularly in forests under restoration. We carried out three independent reciprocal litter transplant experiments to test how litter quality, soil nutrient concentrations, and successional stage (age) influenced HFA in tropical riparian forests. These experimental areas formed a wide gradient of soil and litter nutrients, which we used to evaluate the more general hypothesis that HFA varies with dissimilarity in soil nutrients and litter quality. We found that HFA increased with soil nutrient dissimilarity, suggesting that litter translocation uncouples relationships between decomposers and litter characteristics; and with litter N:P, indicating P limitation in this system. We also found negative HFA effects at a site under restoration that presented low decomposer ability, suggesting that forest restoration does not necessarily recover decomposer communities and nutrient cycling. Within each of the independent experiments, the occurrence of HFA effects was limited and their magnitude was not related to forest age, nor soil and litter quality. Our results imply that HFA effects in tropical ecosystems are influenced by litter nutrient limitation and soil nutrient dissimilarity between home and away sites, but to further disentangle major HFA drivers in tropical areas, a gradient of dissimilarity between litter and soil properties must be implemented in future experimental designs.     

Long-term treatment with fluoxetine induces desensitization of 5-HT4 receptor-dependent signalling and functionality in rat brain

The mode of action of antidepressant drugs may be related to mechanisms of monoamines receptor adaptation, including serotonin 5-HT₄ receptor subtypes. Here we investigated the effects of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine for 21 days (5 and 10 mg/kg, p.o., once daily) on the sensitivity of 5-HT₄ receptors by using receptor autoradiography, adenylate cyclase assays and extracellular recording techniques in rat brain. Fluoxetine treatment decreased the density of 5-HT₄ receptor binding in the CA1 field of hippocampus as well as in several areas of the striatum over the doses of 5–10 mg/kg. In a similar way, we found a significant lower response to zacopride-stimulated adenylate cyclase activity in the fluoxetine 10 mg/kg/day treated group. Furthermore, post-synaptic 5-HT₄ receptor activity in hippocampus-measured as the excitatory action of zacopride in the pyramidal cells of CA1 evoked by Schaffer collateral stimulation was attenuated in rats treated with both doses of fluoxetine. Taken together, these results support the concept that a net decrease in the signalization pathway of 5-HT₄ receptors occurs after chronic selective serotonin reuptake inhibitor treatment: this effect may underlie the therapeutic efficacy of these drugs.     

Holistic assessment of the microbiome dynamics in the substrates used for commercial champignon (Agaricus bisporus) cultivation

Microorganisms strongly influence and are required to generate the selective substrate that provides nutrients and support for fungal growth, and ultimately to induce mushroom fructification under controlled environmental conditions. In this work, the fungal and bacterial microbiota living in the different substrates employed in a commercial crop (compost phase I, II and III, flush 1 and 2, and casing material on day 1, 6 and 8 after compost casing and during flush 1 and 2) have been characterized along the different stages of cultivation by metataxonomic analysis (16S rRNA and ITS2), analysis of phospholipid fatty acid content (PLFAs) and RT-qPCR. Additionally, laccase activity and the content of lignin and complex carbohydrates in compost and casing have been quantified. The bacterial diversity in compost and casing increased throughout the crop cycle boosted by the connection of both substrates. As reflected by the PLFAs, the total living bacterial biomass appears to be negatively correlated with the mycelium of the crop. Agaricus bisporus was the dominant fungal species in colonized substrates, displacing the pre-eminent Ascomycota, accompanied by a sustained increase in laccase activity, which is considered to be a major product of protein synthesis during the mycelial growth of champignon. From phase II onwards, the metabolic machinery of the fungal crop degrades lignin and carbohydrates in compost, while these components are hardly degraded in casing, which reflects the minor role of the casing for nourishing the crop. The techniques employed in this study provide a holistic and detailed characterization of the changing microbial composition in commercial champignon substrates. The knowledge generated will contribute to improve compost formulations (selection of base materials) and accelerate compost production, for instance, through biotechnological interventions in the form of tailored biostimulants and to design environmentally sustainable bio-based casing materials.     

Brevipalpus mites associated with coffee plants (Coffea arabica and C. canephora) in Chiapas,Mexico

Experimental and Applied Acarology - Tenuipalpid mites of the genus Brevipalpus are of significant economic and quarantine importance in agriculture. They can damage and vector phytopathogenic...     

Bax induces cytochrome c release by multiple mechanisms in mitochondria from MCF7 cells

Bax, a pro-apoptotic member of the Bcl-2 family of proteins has the ability to form transmembrane pores large enough to allow cytochrome c (Cyt c) release, as well as to activate the mitochondrial permeability transition pore (mPTP); however, no differential study has been conducted to clarify which one of these mechanisms predominates over the other in the same system. In the present study, we treated isolated mitochondria from MCF7 cells with recombinant protein Bax and tested the efficacy of the mPTP inhibitor cyclosporin A (CsA) and of the Bax channel blocker (Bcb) to inhibit cytochrome c release. We also, induced apoptosis in MCF7 cell cultures with TNF-α plus cycloheximide to determine the effect of such compounds in apoptosis induction via mPTP or Bax oligomerization. Cytochrome c release was totally prevented by CsA and partially by Bcb when apoptosis was induced with recombinant Bax in isolated mitochondria from MCF7 cells. CsA increased the number of living cells in cell culture, as compared with the effect of Bax channel blocker. These results indicate that mPTP activation is the predominant pathway for Bax-induced cytochrome c release from MCF7 mitochondria and for apoptosis induction in the whole cell.