Nucleobindin Co-Localizes and Associates with Cyclooxygenase (COX)-2 in Human Neutrophils

The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis

Impact of Anti-Inflammatory Agents on the Gene Expression Profile of Stimulated Human Neutrophils: Unraveling Endogenous Resolution Pathways

Adenosine, prostaglandin E2, or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A2A receptor agonist CGS 21680, prostaglandin E2, cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution

TRPA1 Contributes to the Acute Inflammatory Response and Mediates Carrageenan-Induced Paw Edema in the Mouse

Transient receptor potential ankyrin 1 (TRPA1) is an ion channel involved in thermosensation and nociception. TRPA1 is activated by exogenous irritants and also by oxidants formed in inflammatory reactions. However, our understanding of its role in inflammation is limited. Here, we tested the hypothesis that TRPA1 is involved in acute inflammatory edema. The TRPA1 agonist allyl isothiocyanate (AITC) induced inflammatory edema when injected intraplantarly to mice, mimicking the classical response to carrageenan. Interestingly, the TRPA1 antagonist HC-030031 and the cyclo-oxygenase (COX) inhibitor ibuprofen inhibited not only AITC but also carrageenan-induced edema. TRPA1-deficient mice displayed attenuated responses to carrageenan and AITC. Furthermore, AITC enhanced COX-2 expression in HEK293 cells transfected with human TRPA1, a response that was reversed by HC-030031. This study demonstrates a hitherto unknown role of TRPA1 in carrageenan-induced inflammatory edema. The results also strongly suggest that TRPA1 contributes, in a COX-dependent manner, to the development of acute inflammation

Lipoteichoic acid-antilipoteichoic acid complexes induce superoxide generation by human neutrophils

Human neutrophils (PMNs) which have been incubated with lipoteichoic acid (LTA) from group A streptococci generated large amounts of Superoxide (O 2 − chemiluminescence and hydrogen peroxide when challenged with anti-LTA antibodies. Cytochalasin B further enhanced O 2 * generation. The onset of Of generation by the LTA-anti-LTA complexes was much faster than that induced by BSA-anti-BSA complexes. LTA-treated PMNs generated much less O 2 * when challenged with BSA complexes, suggesting that LTA might have blocked, nonspecifically, some of the Fc receptors on PMNs. PMNs treated with LTA-anti-LTA complexes further interacted with bystander nonsensitized PMNs resulting in enhanced Of generation, suggesting that small numbers of LTA-sensitized PMNs might recruit additional PMNs to participate in the generation of toxic oxygen species. Protelolytic enzyme treatment of PMNs further enhanced the generation of O 2 − by PMNs treated with LTA-anti-LTA. Superoxide generation could also be induced when PMNs and anti-LTA antibodies interacted with target cells (fibroblasts, epithelial cells) pretreated with LTA. This effect was also further enhanced by pretreatment of the target cells with proteases. PMNs incubated with LTA released lysosomal enzymes following treatment with anti-LTA antibodies. The amounts of phosphatase, Β -glucoronidase, N -acetylglucosaminidase, mannosidase, and lysozyme release by LTA-anti-LTA complexes were much smaller than those released by antibody or histone-opsonized streptococci, suggesting that opsonized particles are more efficient lysosomal enzyme releasers. However, since the amounts of O 2 − generated by the LTA complexes equaled those generated by the opsonized particles, it is assumed that the signals for triggering a respiratory burst and lysosomal enzyme secretion might be different.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44498/1/10753_2004_Article_BF00914316.pd

Cationic polyelectrolytes: A new look at their possible roles as opsonins, as stimulators of respiratory burst in leukocytes, in bacteriolysis, and as modulators of immune-complex diseases (A review hypothesis)

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44497/1/10753_2004_Article_BF00915991.pd

Time-dependent inhibition of oxygen radical induced lung injury

Experimental acute lung injury mediated by reactive metabolites of oxygen can be inhibited by the antioxidant enzymes catalase and Superoxide dismutase (SOD). However, the specific time interval during which these enzymes must be present in order to cause protection is not well defined. Using two experimental models of oxidant-dependent acute lung injury, one involving the intratracheal injection of glucose, glucose oxidase, and lactoperoxidase and the other involving the intravenous injection of cobra venom factor (CVF), we investigated the effects of delaying antioxidant administration on the outcome of the inflammatory response. In both cases, the protective effects of catalase and SOD were rapidly attenuated when their administration was delayed for a short period of time. For example, intratracheal catalase resulted in 98% protection when given simultaneously with the glucose oxidase and lactoperoxidase, but only 13% protection when the catalase was delayed 4 min. Likewise, in the CVF-induced lung injury model, intravenous catalase resulted in 40% protection when given simultaneously with the CVF, but only 2% protection when the catalase was delayed 20 min, even though the peak of the injury occurred hours after the initiation of the injury. A similar time dependence was seen with SOD. These results indicate that antioxidant therapy is required early in the course of oxygen radical-mediated acute lung injury for effective protection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44504/1/10753_2004_Article_BF00914272.pd

A FLOW-SHEET APPROACH TO THE ISSUE OF PYRITE-RICH TAILINGS

The present work is meant to evaluate the effect of separation of pyrite-rich fractions of tailings for both economic and environmental benefit. The project was focused on two tailing dumps in the former mining and processing site of Reps (Northern Albania). In-situ and laboratory investigations were conducted (i) to outline the main environmental characters of the selected dumps with an emphasis on heterogeneities within and between dumps and (ii) to evaluate the concentration potential of commodities, namely pyrite, from the waste materials. Mineralogical and geochemical data were collected and the Acid-Base account procedure was performed in order to evaluate the Acid Drainage (AD) potential of the selected tailing dumps. A quantitative flowsheet was devised in order to relate quantitatively the available information on processing and the collected data. Gravity separation tests were performed by a water shaking table method and the re-processing potential was evaluated through mineralogical and geochemical analyses. The site is subject to long-lasting (103 years) AD processes with significant release of Potentially Toxic Elements into the local environment. Two different classes of tailings have been recognized, based on the sulfide S content. The high- sulfide (S>10%) content material (hS) accounts for about 82% of the total potential H2SO4 production, despite it represents less than 20% of the whole tailing discard. On the contrary, the remnant low- sulfide (S<3%) content material (lS) is the most abundant fraction, yet responsible of a much lower environmental footprint. The two types of tailings are interpreted as by-products of two different mineral processing chains. A single-stage flotation stream yielded to discard lS+hS tailings. On the other hand, a double-stage flotation chain, when active, included a secondary flotation of the tailings produced by the primary Cu flotation. The latter flowsheet yielded to obtain a pyrite concentrate and to discard lS materials. The S content of tailings was thus reduced by over 50%. Re-processing via gravity concentration is an effective method for pyrite recovery from tailings. Tabling tests recovered 29 to 52% of the total sulfide S of the R2 dump, the hS material reworking being the most cost-effective procedure. The final concentrate may also become a sealable product and the gold content may increase the value of pyritic concentrates, given a measured selection of the separation input and parameters. Separation can therefore be used to both confer value to wastes and create smaller sulfide-rich dumps, with lower reclamation costs

Evaluation of heterogeneous sulfide : rich mine tailings as secondary raw materials: a case study in the Mirdita District (Northern Albania)

Sulphide-rich tailings constitute a major environmental concern due to the acid drainage (AD) production and release of potentially toxic elements (PTE). Therefore, the management of active mine dumps and the reclamation of inactive ones require to reduce the exposure of the possible environmental receptors to contamination. A common, critical feature of mine dumps is the heterogeneity of the tailing materials that may complicate the quantitative prediction of potential contamination. On the other hand, heterogeneity can be a key for a more rational and cost-effective remediation, given a good characterization of the dump materials. Moreover, mine wastes often contain valuable components and therefore a suitable solution is to make positive use of them. In fact, as mining and processing (comminution and concentration) constitute a major cost of the ore mineral cycle, the availability of partly-processed, although low-grade resources may represent an interesting opportunity of exploitation. The present work is a case-study for a quantitative flow-sheet-related approach to the aim of heterogeneous sulphide-rich tailing dumps management. We selected the former processing site of Reps, in the Mirdita District (northern Albania), where we characterized two tailing dumps and assessed the contamination. Moreover, we evaluated the concentration potential of commodities, namely pyrite and gold, from the tailing materials. We analysed the Reps tailing samples in order to evaluate mineralogy, grain size and bulk chemical composition, concentration of PTE, the presence and persistence of AD. The data set splits in two classes, based on the sulphide content. The reconstruction of the former processing flow-sheet allowed us to recognize this pattern as resulting from two distinct processing stages and to quantitatively improve the AD prediction results. We selected a shaking table concentration method for testing separation of pyrite and gold, by taking into consideration the need of reducing both costs and volumes. The effectiveness of the method was verified by monitoring both the feed and the output materials for grain size, geochemical and mineralogical parameters. The optimal grain size of the input samples was obtained through a closed-circuit regrinding and milling method. The concentrate output shows an enrichment in pyrite in all the samples. The concentrates constitute 16-37.4 wt% of the shaking table output, depending on the initial sulphide content of the sample. Au concentration data were gained by Activation Analysis on selected grain size classes, in order to verify the correlation of Au concentration and the particle size distribution. Preliminary data show that in our samples a valuable recovery can be achieved by sieving in order to separate the Au- rich (up to 580 ppb) fine fraction (<38\u3bcm), followed by tabling of the coarser fraction. Thus the Metal Recovery reaches 49.6%. Our results show that information about former processing can provide a useful insight for remediation planning. Moreover, this quantitative approach can lead to a rationale recycling of tailings and thus to a reduction of their volumes and environmental impact

La miniera di Herin : uno spaccato storico e geologico nel paesaggio valdostano

Fin dall\u2019epoca dei suoi insediamenti preromani il territorio valdostano \ue8 stato interessato da attivit\ue0 estrattive di minerali metallici e industriali, che ne hanno trasformato profondamente la geografia in senso lato. In Valle d\u2019Aosta sono oggi presenti decine di siti minerari dismessi o abbandonati, che racchiudono un patrimonio sociale, storico e scientifico considerevole. Alcune amministrazioni territoriali promuovono la caratterizzazione, il recupero e la valorizzazione delle aree minerarie dismesse, con particolare riferimento alla legge regionale 12/2008. La miniera di H\ue9rin, pur non rientrando ancora in un progetto di riqualificazione, rappresenta una notevole testimonianza di archeologia industriale e una potenziale attrattiva paesaggistica e scientifica. Il sito minerario (1600-1700 m s.l.m.) si trova nel territorio del Comune di Champdepraz, sulla sinistra orografica dell\u2019omonimo vallone. Quest\u2019ultimo, solcato dal torrente Chalamy, si sviluppa in direzione est-ovest, sulla destra idrografica della Dora Baltea, nella bassa Valle d\u2019Aosta. Dal punto di vista geologico, l\u2019area \ue8 inquadrabile nel contesto dell\u2019unit\ue0 Zermatt-Saas s.s., parte della porzione inferiore ed eclogitica della Zona Ofiolitica Piemontese. Il giacimento di H\ue9rin si presenta sotto forma di corpi lenticolari di solfuri e minori ossidi, da massivi a disseminati. Le litologie incassanti sono prevalentemente scisti granatiferi, a mica chiara o clorite dominanti, con pi\uf9 o meno quarzo, carbonato e diffusi cloritoide e glaucofane, ma sono descritti in letteratura anche glaucofaniti, anfiboliti, prasiniti (Virgilio 1879, Piepoli 1937, Dal Piaz e Omenetto 1978, Bottino et al. 1975, Castello 1995, Fantone 2012). Per la sua collocazione geologica il deposito \ue8 stato assimilato alle mineralizzazioni a pirite e minerali cupriferi associate a metamorfiti ofiolitiche del complesso mesozoico dei calcescisti con pietre verdi (De benedetti 1965, N. Ill. CARG 2010). Le prime testimonianze di coltivazione del giacimento risalgono al 1703, bench\ue9 non sia escluso che esso fosse noto gi\ue0 in epoca romana, o addirittura preromana (Virgilio 1879). Sfruttato a fasi alterne per pi\uf9 di trecento anni, il giacimento di Herin ha rivestito in passato un ruolo rilevante, contribuendo in gran parte alla produzione regionale di rame e di pirite fino alla chiusura degli impianti, avvenuta nel 1957 (Squarzina 1960, Baretti 1968, Binel 1993, Ciardullo 1994). Tra il 2010 e il 2012 \ue8 stato condotto uno studio su questo sito minerario, con il triplice obiettivo di: \u2022 Documentare lo stato attuale della struttura mineraria e individuarne gli aspetti di maggiore interesse culturale in senso lato \u2022 Ricostruire l\u2019evoluzione della coltivazione del giacimento, dal punto di vista dell\u2019estrazione del minerale, della sua lavorazione in situ e della struttura sotterranea \u2022 Produrre uno studio giacimentologico, con particolare interesse per le caratteristiche associabili al percorso metamorfico delle rocce incassanti. Una ricerca bibliografica preliminare ha consentito di delineare la storia della coltivazione mineraria e di individuare alcuni punti di riferimento per l\u2019esplorazione degli ambienti sotterranei. E\u2019 stata quindi eseguita una serie di ricognizioni nelle parti accessibili del complesso minerario, con raccolta di campioni e di materiale fotografico. Successivamente, si \ue8 proceduto al rilevamento di dettaglio di una galleria di carreggio, utilizzando strumenti per il rilevamento topografico in grotta. L\u2019insieme del materiale raccolto \ue8 stato confrontato con le informazioni ricavate dalla bibliografia storica, allo scopo di individuare le evidenze in situ delle fasi di coltivazione del giacimento. Per lo studio della minerogenesi, oggetto di una tesi di laurea (Fantone 2012), sono stati impiegati metodi di microscopia ottica a luce riflessa sulla paragenesi metallica e a luce trasmessa sulle litologie incassanti, integrandoli con analisi chimiche eseguite mediante microsonda elettronica. La geochimica degli elementi in traccia e, in particolare, la distribuzione di Co, Ni e As nella pirite, \ue8 stata determinante per l\u2019elaborazione del modello. E\u2019 stato possibile inquadrare la formazione primaria della mineralizzazione in un contesto vulcanogenico di litosfera oceanica soggetta a processi idrotermali. Lo studio \ue8 stato focalizzato sulla relazione esistente tra le tessiture e la distribuzione degli elementi in traccia, individuando nel percorso metamorfico di retrocessione alcuni processi che hanno determinato l\u2019attuale configurazione del giacimento (Fantone 2012). L'insieme delle informazioni e dei dati raccolti ha permesso di mettere in luce una fitta interconnessione tra gli aspetti giacimentologici, gli elementi del paesaggio e la storia di un\u2019attivit\ue0 che ha condizionato fortemente la configurazione sociale, economica e ambientale del vallone di Champdepraz e dei territori limitrofi