Impaired Assembly Results in the Accumulation of Multiple HLA-C Heavy Chain Folding Intermediates

AbstractClass I MHC H chains assemble with β2-microglobulin (β2m) and are loaded with peptide Ags through multiple folding steps. When free of β2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, β2m-free H chains with calnexin in β2m-defective cells, and with calreticulin and TAP in β2m-expressing cells. In β2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the α1 domain α helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the α1 domain α helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented

Effect of Short-Time Exposures to Nickel and Lead on Brain Monoamine Oxidase from Danio rerio and Poecilia reticulata

The aim of this work was to verify, in two small size freshwater teleosts Danio rerio and Poecilia reticulata, the effects of short-time exposures (24 and 72 h) to a sublethal dose (500 mu g/L) of nickel and lead, on brain monoamine oxidase (MAO), an important neural enzyme. The 24-h treatment using both metals caused a strong reduction of MAO activity in D. rerio brain, whereas causing a slight MAO activity stimulation in P reticulata brain. The same treatment in both species did not affect the brain MAO mRNA production as showed by RT-PCR. Extending the duration of treatment as far as 72 h, partly (D. rerio) or completely (P. reticulata) reversed the metal effects on brain MAO activity suggesting that mechanisms to neutralize the metals had been activated. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 309-313, 2009

Molecular characterization of monoamine oxidase in zebrafish (Danio rerio)

Monoamine oxidase (MAO) is responsible for the degradation of a number of neurotransmitters and other biogenic amines. In terrestrial vertebrates, two forms of the enzyme, named MAO A and B, were found in which mammals are coded by two similar but distinct genes. In teleosts, the biochemical data obtained so far indicate that enzyme activity is due to a single form, whose sequence, obtained for trout, displays 70% identity with mammal MAO A and B. In this paper, we carried out an investigation of zebrafish MAO (Z-MAO) to shed further light on the nature of the MAO form present in aquatic vertebrates. Sequencing studies have revealed an open reading frame 522-amino-acids long with MW 58.7 kDa, displaying 84% identity with trout MAO and about 70% identity with mammal MAO A and MAO B. Analysis of the sequence and of the predicted secondary structure shows that also in Z-MAO principal domains characterizing the MAOs are present. The domain linking the FAD is very well conserved, while the transmembrane domain sequence linking the enzyme to the external mitochondrial membrane does not appear to be conserved even with respect to trout MAO. Comparison with the amino acids which, according to the human MAO B and rat MAO A models, line the substrate-binding site shows that in Z-MAO, several residues (V172, N173, F200, L327) differ from MAO B but are similar or identical to the corresponding ones present in rat MAO A, as well as in trout MAO. A three-dimensional model is reported of the substrate-binding site of Z-MAO obtained by comparative modeling. Our observations support the hypothesis that the MAO form present in aquatic vertebrates is a MAO A-like form. Experiments performed to test the effect of selective MAO A (clorgyline) and MAO B (deprenyl) inhibitors on the enzyme's activity in liver and brain confirm the presence of a single form of MAO in zebrafish. (C) 2004 Elsevier Inc. All rights reserved

Molecular and cellular responses to short exposure to bisphenols A, F, and S and eluates of microplastics in C. elegans

Bisphenol F (BPF) and bisphenol S (BPS) have been developed as an alternative to bisphenol A (BPA), a well-known endocrine disruptor, leading to their detection in the aquatic environment. In this work, we used the animal model Caenorhabditis elegans to improve our understanding of their potential effects on the biota and the environment. Our findings demonstrated that, after 24 h exposure, all the bisphenols examined increased the number of apoptotic corpses and the expression of the detoxifying enzymes SOD-3 and GST-4, without affecting the ROS levels, while BPA and BPS significantly enhanced DNA fragmentation. Furthermore, similarly to BPA, BPF and BPS did not alter the lifespan through the activation of SEK-1 and SKN-1 pathways. Thus, this study raises the attention of the risks associated with exposure to BPA alternatives. We also examined the effects of microplastic (MP) eluates on C. elegans. Aqueous extracts of weathered microplastic samples, both at high and low degradation state and pellets, have been evaluated for their effects on lifespan, DNA fragmentation, germline apoptosis, and oxidative stress response. Overall, our findings showed that eluates of low degraded plastics exert a greater toxic effect on the nematode C. elegans compared with the aqueous sample of high degraded plastic fragments and pellets

MACROFAUNAL COMMUNITY AS INDICATOR OF SANDY BEACHES ENVIRONMENTAL QUALITY

Ecosystems are under the pressure of complex mixtures of contaminants whose effects are not always simple to assess. Among these, sandy beaches environments are one of the most vulnerable and seriously endangered ecosystems in the world; in recent years have been variously disturbed and damaged by human interventions.Biomarkers, acting as early warning signals of the presence of potentially toxic xenobiotic, are useful tools for assessing effects of these compounds and provide information about their bioavailability. In this context, ecotoxicogenomics provides an attractive approach to study changes after exposure to an environmental stressor; variation in gene expression should be taken into consideration when determining xenobiotic mechanisms of action. Our research program foresees investigations at different levels to unravel the biotic effects associated with environmental perturbations. We hence focus on sandy beaches close to the mouth of rivers flowing on the Central Italian Mediterranean coast with the main goal of providing innovative indicators of environmental stressors. For this purpose, we carried out chemical analyses of sand; these results were coupled with the screening of the transcriptomes of the amphipod Talitrus saltator (Talitridae, Amphipoda), a key species of sandy beach communities. Finally, we are firstly applying a biomarker of genotoxicity in T. saltator to monitor the effects of pollution at a low level of biological organization