68 research outputs found
Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells
No full textThe g-aminobutyric acid transporter (GAT-1) isoform of the g-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney
cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G.,and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668–4674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (DC-GAT)
maintained the original functional activity and apicallocalization, either the removal (DC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic
GAT-1 (GBS chimera) and the monotopic human
nerve growth factor receptor, to the basolateral surface.These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral
localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT
Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus
No full textAbstractThe intracellular localization and activity of the nitric oxide synthase (NOS) isoforms were investigated in rat brown adipocytes. Immunohistochemistry showed cytoplasmic and nuclear staining for the endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms; accordingly, anti-L-citrulline antibody, a marker of NOS activity, immunostained both the cytoplasm and the nucleus. The presence of metabolically active NOS in the nucleus was further confirmed by immunoblotting analyses of subcellular fractions of homogenates from cultured brown adipocytes and by measurements of NOS activity in the cytosol and nucleus. Sympathetic stimulation in vivo (i.e. cold exposure or β3-adrenergic agonist treatment) and in vitro (i.e. noradrenaline treatment of cultured cells) significantly increased both cytosolic and nuclear eNOS and iNOS expression and activities. By contrast, the number of iNOS-positive, but not eNOS-positive, nuclei was significantly lower in the functionally impaired brown fat of genetically obese Zucker fa/fa rats. These data suggest the existence of a noradrenaline-modulated functional NOS system in the nucleus of brown adipocytes
White adipocytes are less prone to apoptotic stimuli than brown adipocytes in rodent
No full textObesity in rodents is related to a functional atrophy of brown adipose tissue (BAT), and consequent impairment of adaptive thermogenesis and increased white adipose tissue (WAT) mass.1 Further to defects in molecular processes of the adaptive thermogenesis, the BAT atrophy may be due to an increased rate of brown fat cell death.2 The expression of tumor necrosis factor (TNF)- is elevated in WAT of a variety of obese animals3 and humans,4 and TNF-receptor 1 (TNFR-1) stimulation induces brown adipocyte apoptosis both in vitro and in vivo.2, 5 Although there is some evidence that apoptosis of white adipocytes may be relevant in certain situations,6, 7 its role in WAT accumulation is not clearly defined. Here, a different sensitivity to apoptotic stimuli is reported in brown and white adipocytes, concomitant with a cell-specific expression of pro- and anti-apoptotic molecules. This difference is evident also in BAT and WAT of rats either subjected to starvation or genetically obese
Prion protein structure is affected by pH-dependent interaction with membranes: A study in a model system
No full textAbstractInteraction of full length recombinant hamster prion protein with liposomes mimicking lipid rafts or non-raft membrane regions was studied by circular dichroism, chemical cross-linking and sucrose gradient ultracentrifugation. At pH 7.0, the protein bound palmitoyloleoylphosphatidylcholine/cholesterol/sphingomyelin/monosialoganglioside GM1 (GM1) ganglioside liposomes but not palmitoyloleoylphosphatidylcholine alone (bound/free=0.33 and 0.01, respectively), maintaining the native α-helical structure and monomeric form. At pH 5.0, though still binding to quaternary mixtures, in particular GM1, the protein bound also to palmitoyloleoylphosphatidylcholine (bound/free 0.35) becoming unfolded and oligomeric. The pH-dependent interaction with raft or non-raft membranes might have implication in vivo, by stabilizing or destabilizing the protein
Ischemic conditions and beta-secretase activation: The impact of membrane cholesterol enrichment as triggering factor in rat brain endothelial cells
No full textAmong harmful conditions damaging the blood-brain barrier, cerebral stroke and reperfusion injuries were proposed as contributing factors to Alzheimer's disease etiology. Indeed it was reported that ischemic conditions promote beta-amyloid peptide production in brain endothelial cells, although implicated mechanisms are yet not fully understood.
Oxidative injury related to ischemia affects membrane-lipids profile by altering their biochemical properties and structural dynamics, which are also believed to play significant role in the amyloid precursor protein processing, suggesting a link between alterations in lipid membrane composition and beta-amyloid peptide production enhancement.
Using brain microvascular endothelial cells, here we demonstrate how oxygen and glucose deprivation followed by normal conditions restoration, mimicking ischemic environment, increases cell cholesterol amount (+20%), reduces membrane fluidity and results in strong activation (+40%) of beta-secretase 1 enzymatic activity. Moreover, we observed an increase of amyloid precursor protein and beta-secretase 1 protein levels with altered localization in non-discrete (Triton X-100 soluble) membrane domains, leading to an enhanced production of amyloid precursor protein beta-carboxyl-terminal fragment. Therefore, lipid alterations induced by oxygen and glucose deprivation enhance beta-secretase 1 activity, favor its proximity to amyloid precursor protein and may concur to increased amyloidogenic cleavage. The latter, represents a detrimental event that may contribute to beta-amyloid homeostasis impairment in the brain and to Alzheimer's disease-related BBB dysfunctions
New isoxazolidinone and 3,4-dehydro-β-proline derivatives as antibacterial agents and MAO-inhibitors: A complex balance between two activities
No full textAmong the different classes of antibiotics, oxazolidinone derivatives represent important drugs, since their unique mechanism of action overcomes commonly diffused multidrug-resistant bacteria. Anyway, the structural similarity of these molecules to monoamino oxidase (MAO) inhibitors, like toloxatone and blefoxatone, induces in many cases loss of selectivity as a major concern. A small library of compounds based on isoxazolidinone and dehydro-β-proline scaffold was designed with the aim to obtain antibacterial agents, evaluating at the same time the potential effects of structural features on MAO inhibitory behaviour. The structural modification introduced in the backbone, starting from Linezolid model, lead to a significant loss in antibiotic activity, while a promising inhibitory effect could be observed on monoamino oxidases. These interesting results are also in agreement with docking experiments suggesting a good binding pose of the synthesized compounds into the pocket of the oxidase enzymes, in particular of MAO-B
Study of the antioxidant effects of coffee phenolic metabolites on c6 glioma cells exposed to diesel exhaust particles
No full textThe contributing role of environmental factors to the development of neurodegenerative diseases has become increasingly evident. Here, we report that exposure of C6 glioma cells to diesel exhaust particles (DEPs), a major constituent of urban air pollution, causes intracellular reactive oxygen species (ROS) production. In this scenario, we suggest employing the possible protective role that coffee phenolic metabolites may have. Coffee is a commonly consumed hot beverage and a major contributor to the dietary intake of (poly) phenols. Taking into account physiological concentrations, we analysed the effects of two different coffee phenolic metabolites mixes consisting of compounds derived from bacterial metabolization reactions or phase II conjugations, as well as caffeic acid. The results showed that these mixes were able to counteract DEP-induced oxidative stress. The cellular components mediating the downregulation of ROS included extracellular signal-regulated kinase 1/2 (ERK1/2), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and uncoupling protein 2 (UCP2). Contrary to coffee phenolic metabolites, the treatment with N-acetylcysteine (NAC), a known antioxidant, was found to be ineffective in preventing the DEP exposure oxidant effect. These results revealed that coffee phenolic metabolites could be promising candidates to protect against some adverse health effects of daily exposure to air pollution
Lipid Reshaping and Lipophagy Are Induced in a Modeled Ischemia-Reperfusion Injury of Blood Brain Barrier
No full textIschemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia
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