Shed vesicles are involved in the release of some leaderless proteins.

Most proteins destined for secretion in the extracellular matrix are characterized by the presence of N-terminal signal peptides which direct their translocation into the endoplasmic reticulum, they are subsequently transferred to the Golgi apparatus and then secreted in the extracellular space. A growing number of secreted proteins, are being identified which, however, lack signal peptides allowing their entrance into the endoplasmic reticulum. They include the inflammatory cytokine interleukin 1b, galactins, macrophage migration inhibitory factor (MIF), acid and basic fibroblast growth factors (FGF-1, FGF-2) and Sphingosine kinase1(SphK-1). These proteins are secreted from the cell by unconventional processes which are the subject of numerous studies. Several types of normal and tumor cells can release in the extracellular medium microvesicles, called esovesicles, which result from budding of their plasma membranes. The vesicle diameter ranges between 100nm and 1000nm, the vesicle composition and function depends on the kind of the cell from which they have been produced. We already reported that FGF-2, a secreted lectin that transmits proangiogenic signals, and which is recognized as a potential oncoprotein able to modulate tumour growth and malignancy (Sorensen et al 2006), is released from SkHep1 cells, and from transfected NIH 3T3 cells through vesicle shedding (Taverna et al.2003). Now we are trying to elucidate the intracellular route followed by the growth factor from the site of synthesis to vesicles budding from the cell membrane. Actin filaments appear to be a binary for this intracellular trafficking. After 6h of treatment with cytocalasine, a drug that interferes with actin polymerization, the amount of vesicles was in fact decreased and FGF-2 clustering in granules localized near the cell surface was avoided. On the contrary no effects were observed when cells were treated with drugs which interfere with microtubule polymerization or de-polymerization. We also observed that FGF-2 granules are not included in lipid-coated vesicles. We are also analyzing the possibility that esovesicles are involved in the secretion of another leader-less signalling protein: Sphingosine kinase1 (SphK1). SphK1 has been shown to regulate a wide variety of cellular processes, including promotion of cell proliferation, survival and motility (Spiegel et al. 2003). SphK1 is primarily localized in the cytosol; when a signal induces the phosphorylation of Ser 225 of SphK1 through the activation of MAPK and ERK1/2, the molecule is translocated in plasma membranes and the involvement of actin filaments in its targeting has been reported (Pitson et. al. 2003). Three SphK1 isoforms having a different number of amino acids (384, 398 and 470) have been identified, we found that extracellular vesicles are enriched in the 47kDa isoform. SphK assays with TLC confirm that the enzyme is present in shed vesicles and that it has enzymatic activity. The substrate Sphingosine is also present in esovesicles therefore shed vesicles are likely to be a site of Sphingosine 1 Phosphate production

Acetylcholine-treated murine dendritic cells promote inflammatory lung injury

In recent years a non-neuronal cholinergic system has been described in immune cells, which is often usually activated during the course of inflammatory processes. To date, it is known that Acetylcholine (ACh), a neurotransmitter extensively expressed in the airways, not only induces bronchoconstriction, but also promotes a set of changes usually associated with the induction of allergic/Th2 responses. We have previously demonstrated that ACh polarizes human dendritic cells (DC) toward a Th2-promoting profile through the activation of muscarinic acetylcholine receptors (mAChR). Here, we showed that ACh promotes the acquisition of an inflammatory profile by murine DC, with the increased MHC II IAd expression and production of two cytokines strongly associated with inflammatory infiltrate and tissue damage, namely TNF-α and MCP-1, which was prevented by blocking mAChR. Moreover, we showed that ACh induces the up-regulation of M3 mAChR expression and the blocking of this receptor with tiotropium bromide prevents the increase of MHC II IAd expression and TNF-α production induced by ACh on DC, suggesting that M3 is the main receptor involved in ACh-induced activation of DC. Then, using a short-term experimental murine model of ovalbumin-induced lung inflammation, we revealed that the intranasal administration of ACh-treated DC, at early stages of the inflammatory response, might be able to exacerbate the recruitment of inflammatory mononuclear cells, promoting profound structural changes in the lung parenchyma characteristic of chronic inflammation and evidenced by elevated systemic levels of inflammatory marker, TNF-α. These results suggest a potential role for ACh in the modulation of immune mechanisms underlying pulmonary inflammatory processes.Fil: Gori, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Alcain, Julieta María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Vanzulli, Silvia. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Moreno Ayala, Mariela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Candolfi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Jancic, Carolina Cristina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Cátedra de Microbiología, Parasitología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Cátedra de Microbiología, Parasitología e Inmunología; ArgentinaFil: Vermeulen, Elba Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Cátedra de Microbiología, Parasitología e Inmunología; ArgentinaFil: Salamone, Gabriela Veronica. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Cátedra de Microbiología, Parasitología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis

Applicazione di un veloce metodo di estrazione del DNA per l’identificazione di prodotti ittici marini.

L'identificazione dei prodotti ittici è uno dei temi chiave in materia di sicurezza alimentare. L’errata etichettatura dei prodotti alimentari e la sostituzione di alcuni ingredienti rappresentano questioni emergenti in termini di qualità e sicurezza alimentare e nutrizionale. L'autenticazione e la tracciabilità dei prodotti alimentari, gli studi di tassonomia e di genetica di popolazione, così come l'analisi delle abitudini alimentari degli animali e la selezione delle prede, si basano su analisi genetiche tra cui la metodica molecolare del DNA barcoding, che consiste nell’amplificazione e nel sequenziamento di una specifica regione del gene mitocondriale chiamata COI. Questa tecnica biomolecolare è utilizzata per fronteggiare la richiesta di determinazione specifica e/o la reale provenienza dei prodotti commercializzati, nonché per smascherare errori di etichettatura e sostituzioni fraudolente, difficile da rilevare soprattutto nei prodotti ittici trasformati. Sul mercato sono disponibili differenti kit per l'estrazione del DNA da campioni freschi e conservati; l’impiego dei kit, aumenta drasticamente il costo dei progetti di caratterizzazione e di genotipizzazione dei campioni da analizzare. In questo scenario è stato messo a punto un metodo veloce di estrazione del DNA. Esso non prevede nessuna fase di purificazione per i prodotti ittici freschi e trasformati e si presta a qualsiasi analisi che preveda l’utilizzo della tecnica PCR. Il protocollo consente l'amplificazione efficiente del DNA da qualsiasi scarto industriale proveniente dalla lavorazione del pesce, indipendentemente dal metodo di conservazione del campione. L’applicazione di questo metodo di estrazione del DNA, combinato al successo e alla robustezza della amplificazione PCR (secondo protocollo barcode) ha permesso di ottenere, in tempi brevissimi e con costi minimi, il sequenziamento del DNA

Papel dual de la linfopoyetina estromal tímica (TSLP): ¿regulador homeostático o mediador pro-inflamatorio?

Linfopoyetina estromal tímica o TSLP es una citoquina emparentada con la IL-7, producida principalmente por células epiteliales del pulmón, piel e intestino. La TSLP fue originalmente descripta por activar fuertemente a las células dendríticas mieloides, induciendo una respuesta Th2 inflamatoria caracterizada por alta producción de TNF- α y poca o nula producción de IL-10, diferenciándose de las respuestas Th2 regulatorias caracterizadas por una baja producción de TNF- α y alta producción de IL-10. En los últimos años, se ha descripto una correlación directa entre la expresión de TSLP por el epitelio y la patogénesis de enfermedades tales como la dermatitis atópica y el asma, al observarse una elevada expresión de esta citoquina en queratinocitos de lesiones cutáneas de pacientes con dermatitis atópica y en la mucosa bronquial de pacientes asmáticos. Sin embargo, estudios más recientes sugieren que TSLP también puede desempeñar un papel clave en el desarrollo de un perfil Th2 protector en intestino, siendo crítico para el mantenimiento de la homeostasis y tolerancia de la mucosa intestinal mediante la limitación de las respuestas inmunitariasFil: Gori, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Alcain, Julieta María. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Vermeulen, Elba Monica. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Salamone, Gabriela Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin

The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii

Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257 bp with a 5′-untranslated region (UTR) of 63 bp and a 3′-UTR of 331 bp with a poly (A) tail, and an open-reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33 kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain and a nuclear localization signal, strongly suggesting that PcCNBP was a homolog of vertebrate CNBP. The PcCNBP transcript was constitutively expressed in all tested tissues of unchallenged crayfish, including hepatopancreas, gill, eyestalk, haemocytes, intestine, stomach and cuticle with highest expression in haemocytes, intestine, gills and hepatopancreas. The mRNA expression of PcCNBP in haemocytes was modulated at transcriptional level by different immune challenges, suggesting its involvement in the immune response of P. clarkii during both bacteria and viruses infection

A novel enzyme blend for efficient tissue dissociation and primary cells isolation

Tissue dissociation/primary cell isolation and cell harvesting are principal appli- cations for enzymes in tissue culture research and cell biology studies. The goal of a cell isolation procedure is to maximize the yield of functionally viable dissoci- ated cells. Among the parameters which affect the outcome of any particular dissociating procedure there are enzyme(s) used and related impurities presents in crude enzyme preparation. ABIEL srl recently produced the recombinant collagenase class I (Col G) and II (Col H) from Clostridium histolyticum (PCT WO 2011/073925 A9). The enzymes were produced in Escherichia coli and purified by affinity chromatography. The method of production adopted allows absolute control of the final composition of these enzymes, as well as their stability, purity, activity, absence of toxicity and higher reproducibility of batches. The two collagenases produced separately have been used in conjunction according to precise proportions to dissociate calvaria, liver, pancreas, retina of the BALB/c mouse; and bovine hoof. The analyses carried out on all isolated cell populations suggest that the cells maintain the structural and functional integrity of specific tissues/organs originating. Recombinant Col G and Col H enzymes produced by ABIEL are promising in the context of the tissue/cells dissociation, with the aim to make innovation in the fields of tissue engineering and transplantation medicine

Herbicidal Activity of Thymbra capitata (L.) Cav. Essential Oil

[EN] The bioherbicidal potential ofThymbra capitata(L.) Cav. essential oil (EO) and its main compound carvacrol was investigated. In in vitro assays, the EO blocked the germination and seedling growth ofErigeron canadensisL.,Sonchus oleraceus(L.) L., andChenopodium albumL. at 0.125 mu L/mL, ofSetaria verticillata(L.) P.Beauv.,Avena fatuaL., andSolanum nigrumL. at 0.5 mu L/mL, ofAmaranthus retroflexusL. at 1 mu L/mL and ofPortulaca oleraceaL., andEchinochloa crus-galli(L.) P.Beauv. at 2 mu L/mL. Under greenhouse conditions,T. capitataEO was tested towards the emergent weeds from a soil seedbank in pre and post emergence, showing strong herbicidal potential in both assays at 4 mu L/mL. In addition,T. capitataEO, applied by spraying, was tested againstP. oleracea,A. fatuaandE. crus-galli. The species showed different sensibility to the EO, beingE. crus-gallithe most resistant. Experiments were performed againstA. fatuatestingT. capitataEO and carvacrol applied by spraying or by irrigation. It was verified that the EO was more active at the same doses in monocotyledons applied by irrigation and in dicotyledons applied by spraying. Carvacrol effects onArabidopsisroot morphology were also studied.This research was supported by the Universitat Politècnica de València [project number: SP20120543], by Generalitat Valenciana [project number GV/2014/039], and by the Spanish Ministry of Science, Innovation and Universities [project number: RTI2018¿094716¿B¿I00]. Thanks to Jovano Erris Nugroho and Muhamad Iqbal who collaborate to carry out in vivo experiment 4 during their internship in the Plant Health in Sustainable Cropping Systems Erasmus+ Programme. This research work has been developed as a result of a mobility stay funded by the Erasmus+-KA1 Erasmus Mundus Joint Master Degrees Programme of the European Commission under the PLANT HEALTH Project. 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Tecniche di campionamento di sostanze bioattive da aculei di Scorpaena porcus

Nel presente report vengono descritte le tecniche utilizzate per l'estrazione del veleno dagli aculei della specie ittica Scorpaena porcus. Gli estratti grezzi ottenuti sono successivamente processati al fine di ottenere frazioni da utilizzare in saggi biochimici, per lo studio di possibili attività biologiche