Experiences from the accident of Seveso

Provisional data on selected sanitary events which took place at Seveso after July 10 1976 are reported. 187 cases of chloracne, mostly in children, were detected, 50 just after the accident, the others within a year. Most polluted area (zone A) provided almost all "early" and most severe cases, but the territorial distribution of chloracne prevalence rates showed some inconsistencies with the soil TCDD pollution map; interpretations for such findings are discussed. Thirty-eight birth defects were detected in 1977 (none in zones A and B), more than in previous years, but still less than expected in a well controlled "normal" population: no clustering around a given type was observed. Spontaneous abortions, evaluated both as abortion rates and as pregnancy loss rates, showed scattered and statistically non-significant variations, inconsistent with the pollution map. No differences in birth and death rates compared to surrounding areas were observed. Data on ad hoc cytogenetic, neurological and immunological surveys are commented. Limitations of the presently available data are discussed and further research lines are anticipated.     

Chronic Nicotine Treatment Impacts the Regulation of Opioid and Non-opioid Peptides in the Rat Dorsal Striatum

The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.Nicotine is the main psychoactive ingredient of tobacco (1). By acting on the nicotinic acetylcholine receptors located in diverse brain areas, nicotine generates psychoactive effects such as euphoria, reduced stress, increased energy, and enhanced cognitive functions (2). Chronic nicotine use alters various aspects of neurochemical transmission and has a strong impact on diverse physiological processes (2), resulting in drug-seeking and drug-taking behaviors for normal smokers and for a considerable number of patients suffering from schizophrenia and Alzheimer disease, who use nicotine for self-medication (3, 4). The dorsal striatum (DS)¹ is one of the key brain regions that has been associated with neural regulation during chronic nicotine exposure (5). In particular, the DS is involved in habit formation during the preoccupation/craving (later) phase of nicotine dependence characterized by compulsive drug-taking (6). Behavioral changes associated with nicotine dependence have been linked to small molecule neurotransmitter systems, including the glutamate and dopamine system in the DS (7). The DS is also known to contain diverse neuropeptides, many of which are probably critical mediators of physiological processes that are associated with nicotine, such as the regulation of reinforcement and energy metabolism. However, neuropeptides have not been extensively investigated in the DS during long periods of nicotine administration.Immunoassay studies have shown that neuropeptides, including substance P, neuropeptide Y, and opioid peptides, including the enkephalins, are expressed by inhibitory neurons (8), which make up a large majority of the neurons in the DS (9). Many of these inhibitory GABAergic neurons express nicotinic cholinergic receptors (10), suggesting that nicotine administration may regulate their activity, leading to variations in the release of neuropeptides, as well as the inhibitory neurotransmitter GABA. Previous investigations of peptide regulation during chronic nicotine administration in the striatum have exclusively focused on the class of opioid peptides, which are thought to play an important role in the control of diverse physiological processes, including reward processing, nociception, and regulation of emotions (11, 12). Available studies have focused on the analysis of three opioid peptides, their precursors, or receptors as follows: met-enkephalin, dynorphin, and β-endorphin, using conventional techniques like immunoassays (13, 14). There is considerable variability in reported changes of peptide levels in the striatum during chronic nicotine administration. For example, when animals are treated with 1 mg/kg free base nicotine (daily for 14 days), met-enkephalin increased in the striatum (15). By contrast, met-enkephalin is reduced in the striatum when rats are treated with 0.3 mg/kg nicotine (three times/day for 14 days) (16). A number of factors might contribute to this observed variability, including the exact dosing, daily frequency, time span of administration, and delivery method of nicotine. Furthermore, as individual studies have each so far generally examined a single opioid peptide, there is currently little reliable information about peptide co-regulation, even for these well studied opioid peptides. In addition to these opioid peptides, the DS expresses peptides from other peptide families, which are also potential targets under the regulation of chronic nicotine treatment. So far, however, there is no information available about changes of these non-opioid peptides during chronic nicotine administration.In this study, our aim was to use a neuropeptidomics approach (17) to provide a comprehensive characterization of dorsal striatal neuropeptides after long term nicotine chronic treatment in adult rats using oral administration. The main advantage of this approach is that it allows the simultaneous monitoring of many peptides from the same brain tissue derived from a single drug protocol. We used a combination of a robust sample preparation method (18), high accuracy LC-MS analysis (19, 20), and the use of multiple synthetic internal standards (21) to compare peptide levels in the DS between chronic nicotine and control animals. Our peptidome analysis determined 14 peptides exhibiting significant changes following chronic nicotine administration. Among these peptides were members of the opioid family that had previously been associated with nicotine dependence, as well as a number of newly identified peptides, including members of the secretogranin, cholecystokinin, and somatostatin families. This greatly expands the present scope of peptide involvement in drug dependence in the dorsal striatum.     

Gigantism among Late Jurassic limulids: New ichnological evidence from the Causses Basin (Lozère,France) and comments on body-size evolution among horseshoe crabs

An abundant ichnological material composed of xiphosuran trackways and isolated traces was discovered in Upper Jurassic limestones from the Causses Basin (Causse Méjean, Lozère, France). The morphology of the imprints supports their identification as Kouphichnium isp. In contrast to the most frequent case, the trackways are composed of omnipresent pusher imprints sometime associated with leg traces, but with no telson mark. We argue that this pattern reflects actual surface traces rather than an incomplete set of undertracks. The size distribution of the sampled ichnites is broadly bimodal. This is best explained by sexual dimorphism, a phenomenon frequently observed in modern xiphosurans. Analysis of the trace fossils further suggests that several growth stages are recorded and that the horseshoe crabs were walking in a protected and flat environment like a lagoon. This area, certainly close to a mating ground, was occasionally affected by a continental influence. The biometric study of the tracks suggests a gigantic size for the trackmakers whose body may have reached 84 cm in length. This discovery complements the few reports on other gigantic horseshoe crabs in the Jurassic of Western Europe, thus casting doubt on the postulated increase in body size from the Palaeozoic to the Recent. Furthermore, a literature review shows that there are still major gaps in the record of limulid body-fossils and tracks. Thus, neither of these archives can be taken at face value for quantifying the body-size evolution of horseshoe crabs.     

Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli

The biogenesis of biological membranes hinges on the coordinated trafficking of membrane lipids between distinct cellular compartments. The bacterial outer membrane enzyme PagP confers resistance to host immune defenses by transferring a palmitate chain from a phospholipid to the lipid A (endotoxin) component of lipopolysaccharide. PagP is an eight-stranded antiparallel beta-barrel, preceded by an N-terminal amphipathic alpha-helix. The active site is localized inside the beta-barrel and is aligned with the lipopolysaccharide-containing outer leaflet, but the phospholipid substrates are normally restricted to the inner leaflet of the asymmetric outer membrane. We examined the possibility that PagP activity in vivo depends on the aberrant migration of phospholipids into the outer leaflet. We find that brief addition to Escherichia coli cultures of millimolar EDTA, which is reported to replace a fraction of lipopolysaccharide with phospholipids, rapidly induces palmitoylation of lipid A. Although expression of the E. coli pagP gene is induced during Mg2+ limitation by the phoPQ two-component signal transduction pathway, EDTA-induced lipid A palmitoylation occurs more rapidly than pagP induction and is independent of de novo protein synthesis. EDTA-induced lipid A palmitoylation requires functional MsbA, an essential ATP-binding cassette transporter needed for lipid transport to the outer membrane. A potential role for the PagP alpha-helix in phospholipid translocation to the outer leaflet was excluded by showing that alpha-helix deletions are active in vivo. Neither EDTA nor Mg(2+)-EDTA stimulate PagP activity in vitro. These findings suggest that PagP remains dormant in outer membranes until Mg2+ limitation promotes the migration of phospholipids into the outer leaflet.     

Prunasin hydrolases during fruit development in sweet and bitter almonds

Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the β-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet.     

High identification rates of endogenous neuropeptides from mouse brain

Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 μL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC-MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.     

Extensive characterization of Tupaia belangeri neuropeptidome using an integrated mass spectrometric approach

Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed, and targeted liquid chromatography (LC)-Fourier transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search, and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have sequences identical or similar to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.     

Separation and identification of mouse brain tissue microproteins using top‐down method with high resolution nanocapillary liquid chromatography mass spectrometry

Microproteins and endogenous peptides in the brain contain important substances that have critical roles in diverse biological processes, contributing to signal transduction and intercellular signaling. However, variability in their physical or chemical characteristics, such as molecule size, hydrophobicity, and charge states, complicate the simultaneous analysis of these compounds, although this would be highly beneficial for the field of neuroscience research. Here, we present a top‐down analytical method for simultaneous analysis of microproteins and endogenous peptides using high‐resolution nanocapillary LC‐MS/MS. This method is detergent‐free and digestion‐free, which allows for extracting and preserving intact microproteins and peptides for direct LC‐MS analysis. Both higher energy collision dissociation and electron‐transfer dissociation fragmentations were used in the LC‐MS analysis to increase the identification rate, and bioinformatics tools ProteinGoggle and PEAKS Studio software were utilized for database search. In total, we identified 471 microproteins containing 736 proteoforms, including brain‐derived neurotrophic factor and a number of fibroblast growth factors. In addition, we identified 599 peptides containing 151 known or potential neuropeptides such as somatostatin‐28 and neuropeptide Y. Our approach bridges the gap for the characterization of brain microproteins and peptides, which permits quantification of a diversity of signaling molecules for biomarker discovery or therapy diagnosis in the future.