Reactivated memories compete for expression after Pavlovian extinction

We view the response decrement resulting from extinction treatment as an interference effect, in which the reactivated memory from acquisition competes with the reactivated memory from extinction for behavioral expression. For each of these memories, reactivation is proportional to both the strength of the stimulus-outcome association and the quality of the facilitatory cues for that association which are present at test. Here we review basic extinction and recovery-from-extinction phenomena, showing how these effects are explicable in this associative interference framework. Moreover, this orientation has and continues to dictate efficient manipulations for minimizing recovery from extinction. This in turn suggests procedures that might reduce relapse from exposure therapy for a number of psychological disorders. Some of these manipulations enhance the facilitatory cues from extinction that are present at test, others strengthen the extinction association (i.e., CS-no outcome), and yet others seem to work by a combination of these two processes.     

Hypoxic regulation of angiotensin-converting enzyme 2 and Mas receptor in human CD34+ cells

CD34⁺ hematopoietic stem/progenitor cells (HSPCs) are vasculogenic and hypoxia is a strong stimulus for the vasoreparative functions of these cells. Angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1–7)/Mas receptor (MasR) pathway stimulates vasoprotective functions of CD34⁺ cells. This study tested if ACE2 and MasR are involved in the hypoxic stimulation of CD34⁺ cells. Cells were isolated from circulating mononuclear cells derived from healthy subjects (n = 46) and were exposed to normoxia (20% O₂) or hypoxia (1% O₂). Luciferase reporter assays were carried out in cells transduced with lentivirus carrying ACE2- or MasR- or a scramble-3′-untranslated region gene with a firefly luciferase reporter. Expressions or activities of ACE, angiotensin receptor Type 1 (AT1R), ACE2, and MasR were determined. In vitro observations were verified in HSPCs derived from mice undergoing hindlimb ischemia (HLI). In vitro exposure to hypoxia-increased proliferation and migration of CD34⁺ cells in basal conditions or in response to vascular endothelial growth factor (VEGF) or stromal-derived factor 1α (SDF) compared with normoxia. Expression of ACE2 or MasR was increased relative to normoxia while ACE or AT1R expressions were unaltered. Luciferase activity was increased by hypoxia in cells transfected with the luciferase reporter plasmids coding for the ACE2- or MasR promoters relatively to the control. The effects of hypoxia were mimicked by VEGF or SDF under normoxia. Hypoxia-induced ADAM17-dependent shedding of functional ACE2 fragments. In mice undergoing HLI, increased expression/activity of ACE2 and MasR were observed in the circulating HSPCs. This study provides compelling evidence for the hypoxic upregulation of ACE2 and MasR in CD34⁺ cells, which likely contributes to vascular repair.     

Biofilter efficiency of <i>Eichhornia crassipes</i> in wastewater treatment of fish farming in Amazonia

Fish is a very important part of the human diet in Amazonia. Near the growing cities, fish populations and individual size have decreased over the past decades. Alternatives to traditional and industrial fishing arise, including fish farming. Strategies to minimize the impact of fish farms on the environment are needed to have a regular and healthy fish supply. This is to avoid a reduction of biodiversity, a depletion of natural resources, and/or the induction of significant changes in the structure and functioning of adjacent ecosystems. Very little research has been performed on management of effluents as to maintain the quality of water resources. The present study aimed at testing the efficiency of the Amazonian aquatic macrophyte Eichhornia crassipes as a biofilter for the treatment of effluents from fish farming. In three filtering treatments (50%, 75% and 100% plant cover) and a control (0%), physical and chemical properties of the water were measured and analyzed in a nursery with fish after passing the biofilter system, with a hydraulic retention time of 24 hours. The analyzed variables showed no significant differences (p>0.05) among the treatments with 50-100% cover, indicating that 50% cover would be enough for a good efficiency of the biofilter. All parameters were reduced after passage of the biofilter under the presence of E. crassipes: 73.7% for electrical conductivity, 15% for pH, 84.5% for turbidity, 86.8% for nitrite, 69% for total phosphorus, and 77.8% for orthophosphate. The concentrations of total nitrogen, nitrate and ammonium ions were not significantly changed (p>0.05). We conclude that E. crassipes is effective in improving the quality of effluents from fish farming, with less efficiency for nitrogen compounds. Our treatment system can be adopted by small and medium-sized farmers, aiming at a sustainable employment of the activity.     

Ability to grow on lipids accounts for the fully virulent phenotype in neutropenic mice of Aspergillus fumigatus null mutants in the key glyoxylate cycle enzymes

Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifungals to treat patients suffering from this disease. Fungal proteins playing a crucial role in pathogenesis and with no orthologue in human cells are considered as primary therapeutic targets for the development of new antifungals with a high therapeutic index, one of the major drawbacks of the standard antifungal therapy, so far. In this work, we have analyzed the role in pathogenesis of the key enzymes of the Aspergillus fumigatus glyxoxylate cycle, isocitrate lyase and malate synthase, two possible candidates to primary therapeutic targets in this fungus. Deletion strains lacking isocitrate lyase (DeltaacuD strains) or malate synthase (DeltaacuE mutants) were constructed in this work. The Neurospora crassa pyr-4 gene was used as the replacing marker in gene deletion experiments. The pathogenicities of DeltaacuD and DeltaacuE mutants were tested in neutropenic mice and compared with those of two reference wild-type isolates A. fumigatus 237 and A. fumigatus 293. Interestingly, virulence and cytological studies clearly indicated the dispensability of the A. fumigatus glyoxylate cycle for pathogenicity. In addition, these results suggested the suitability of the pyr-4 gene as a valuable replacing marker for virulence studies in this fungus, a fact that was further confirmed by gene expression analyses. Finally, growth tests were performed to investigate the germination and growth of the DeltaacuD and DeltaacuE strains in nutrient deprivation environments, resembling the conditions that A. fumigatus conidia face after phagocytosis. Results obtained in this work strongly suggest that the ability to grow on lipids (triglycerides) of A. fumigatus isocitrate lyase and malate synthase deletion strains accounts for their fully virulent phenotype.     

Adipocyte differentiation is modulated by secreted delta-like (dlk) variants and requires the expression of membrane-associated dlk

Previous studies demonstrate that the delta-like (dlk) and preadipocyte factor 1 (Pref-1) genes encode similar proteins. Pref-1 is downregulated during adipocyte differentiation, and expression of ectopic Pref-1 inhibits adipogenesis. We explored whether dlk functions similarly to Pref-1 and studied the role of alternately spliced dlk variants encoding membrane-associated or -secreted forms. We also studied whether enforced downregulation of dlk/Pref-1 may enhance the differentiation response of non-committed cells. Ectopic expression of a potentially secreted dlk variant, conditioned media from dlk expressing cells or several individual epidermal-growth-factor-dlk peptides inhibited 3T3-L1 differentiation. This demonstrates that dlk and Pref-1 are functionally equivalent. dlk gene mRNA encoding for secreted variants decreased much faster than total dlk gene mRNA during differentiation of 3T3-L1 cells. In fact, total dlk or membrane-associated dlk protein expression increased during the first hours of differentiation. Cells sorted for lowest levels of dlk protein diminished or lost their ability to differentiate. These data suggest that membrane and secreted dlk protein variants play opposite roles in the control of adipogenesis. In addition, enforced downregulation of dlk protein expression in the weakly adipogenic Balb/c 3T3 cell line dramatically enhanced adipogenesis in response to insulin. These results indicate that dlk protein not only participates in processes leading to inhibition of adipogenesis but that the control of its expression and different spliced variants is essential for the adipogenic response to extracellular signals.     

Functional analysis of mutations in the human carnitine/acylcarnitine translocase in Aspergillus nidulans

Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid beta-oxidation, is usually lethal in both humans and animals, precluding the development of animal models of the disease. In contrast, CACT deficiency is conditionally lethal in the fungus Aspergillus nidulans, since loss-of-function mutations in acuH, the translocase structural gene, do not prevent growth on carbon sources other than ketogenic compounds, such as fatty acids. Here, we describe the molecular characterization of extant acuH alleles and the development of a fungal model for CACT deficiency based on the ability of human CACT to fully complement, when expressed at physiological levels, the growth defect of an A. nidulans DeltaacuH strain on acetate and long-chain fatty acids. By using growth tests and in vitro assays this model enabled us to carry out a functional characterization of human CACT mutations showing that it may be useful for distinguishing potentially pathogenic human CACT missense mutations from neutral, single residue substitution-causing polymorphisms.     

dlk modulates mitogen-activated protein kinase signaling to allow or prevent differentiation

The EGF-like membrane protein dlk plays a crucial role in the control of cell differentiation. Overexpression of the protein prevents, whereas inhibition of its expression increases, adipocyte differentiation of 3T3-L1 cells in response to Insulin-like Growth Factor I (IGF-1) or insulin. We have investigated whether dlk modulates the signaling pathways known to control this process. We found that the levels of dlk expression modulated signaling through the IGF-1 receptor, causing changes in the activation levels and kinetics of Extracellular-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) that correlated with differentiation outcome. These changes occurred in response to IGF-1 or insulin but not in response to Epidermal Growth Factor. However, the levels of expression of IGF-1 receptor, or the activation of Insulin Receptor Substrate-1 in response to IGF-1, were not affected by the levels of dlk expression. Therefore, dlk appears to modulate ERK/MAPK signaling in response to specific differentiation signals.     

The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes

In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.     

The novel gene EGFL9/Dlk2, highly homologous to Dlk1, functions as a modulator of adipogenesis

The Dlk1 gene appears to function as a regulator of adipogenesis. Adult Dlk1-deficient mice are obese, but adipose tissue still develops in transgenic mice overexpressing an Fc-dlk1 fusion protein, and neither type of genetically modified mice displays serious abnormalities. It was therefore possible that one yet unidentified gene might either compensate or antagonize for the absence or for overexpression, respectively, of Dlk1 in those animals. In database searches, we found a novel gene, EGFL9, encoding for a protein whose structural features are virtually identical to those of dlk1, suggesting it may function in a similar way. As dlk1 does, the protein encoded by EGFL9/Dlk2 affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells; however, it does so in an opposite way to that of dlk1. In addition, expression levels of both genes appear to be inversely correlated in both cell lines. Moreover, enforced changes in the expression of one gene affect the expression levels of the other. Our data suggest that adipogenesis may be modulated by the coordinated expression of Dlk1 and EGFL9/Dlk2.     

The EGF-like protein dlk1 inhibits notch signaling and potentiates adipogenesis of mesenchymal cells

The EGF-like homeotic gene Dlk1 appears to function as an inhibitor of adipogenesis. Overexpression of Dlk1 prevents adipogenesis of 3T3-L1 cells. Dlk1-deficient mice are obese; however, adipose tissue still develops in Fc-dlk1 transgenic mice, suggesting that Dlk1 is not a strict inhibitor of adipogenesis. To clarify the role of Dlk1 in adipogenesis, we studied whether Dlk1 could act differently on this process depending upon the differentiation state of the precursor cells. We found that Dlk1 is a potentiator of adipogenesis for mesenchymal C3H10T1/2 cells. This potentiating effect can be triggered by overexpressing the entire protein or the extracellular EGF-like-containing region, but not by overexpressing the intracellular dlk1 sequence. In addition, coculture of C3H10T1/2 cells with other cells expressing Dlk1, but not with cells lacking Dlk1 expression, enhances their adipogenic response. Potentiation of adipogenesis by Dlk1 was associated with changes in the activation of ERK1/2 after IGFI/insulin induction. Finally, as reported with other cells, dlk1 functioned as a Notch signaling inhibitor in C3H10T1/2 cells, but inhibition of Notch1 expression prevented the potentiating effects of Dlk1 in adipogenesis. These data suggest that Dlk1 may potentiate or inhibit adipogenesis depending upon the cellular context, and that Notch1 expression and activation are important factors in this context.