Identification and isolation of primary cilia in the HAT-7 cell line

Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2015 (Department of Molecular and Cell Biology).Includes bibliographic references: leaves 44-48.Primary cilia (PC) are 9+0 arranged microtubule based cellular organelles that are present on the surface of most eukaryotic cells. Their functions in various locations are not fully understood but current data is suggestive of important mechano- and chemo-sensory roles. More specifically PC have been linked to anterior-posterior limb patterning, endochondral bone formation, maintenance of bone, craniofacial development and tooth development. The electron microscopic presence of PC on odontoblasts and their dynamic expression on differentiating ameloblasts suggests possible non-elucidated signaling pathways. In the rat incisor tooth model, development and eruption occur continuously most likely from the cervical loop which has been labeled as the stem cell niche . The labial surface of the developing rat incisor contains a gradation of different stage cells from the progenitor cells of the cervical loop at the apex to the maturation stage ameloblasts below completely mineralized enamel. In this experiment attempts were made to identify and isolate samples of PC without non-ciliary contaminants from HAT-7 cell cultures . Protein isolates of the cytosol, membrane, nuclear and cytoskeleton of HAT-7 and IMCD3 cells were formed using fractionation techniques. All samples were examined using biochemical characterization via SOS-PAGE ... [TRUNCATED

Natura o cultura? L’antropologia della letteratura tra Wolfgang Iser e Darwinismo letterario

La ricerca iseriana di un’antropologia della letteratura cerca di innestare la riflessione sulla narrazione in un campo multidisciplinare molto più ampio del tradizionale settore della critica. Concentrandosi sui concetti di fiction e di fictionalizing, di finzione e del finzionalizzare, Iser prova a mostrare il carattere strumentale della letteratura, intesa quale dispositivo universale d’interpretazione e di creazione di significato.Non c'è dubbio che l’idea iseriana della letteratura come grimaldello esperienziale abbia trovato terreno fertile negli Stati Uniti, seppur in maniera indiretta e probabilmente solo coincidenziale. Qui, infatti, grazie soprattutto a Joseph Carroll, è nato e si è diffuso il cosiddetto Literary Darwinism che, come Iser, ritiene che la letteratura – e quindi la finzione – sia un tratto radicalmente caratteristico dell’essere umano. Altrettanto chiaro però è che le assonanze tra le due visioni finiscono qui, almeno nella misura in cui è possibile riscontrare una prima divergenza sul modo in cui esse intendono il termine “finzione”: mentre per Iser, infatti, la finzione è limitata esclusivamente all’ambito letterario, per i darwinisti letterari il termine arriva a includere un macrocosmo di fenomeni – tra i quali, ad esempio, il sogno e le finzioni del “quotidiano”, due realtà che Iser, viceversa, esclude dal regno finzionale.Ora, è innanzitutto su questo punto di divergenza che il presente contributo intende riflettere, sforzandosi, in primo luogo, di mettere in evidenza le diverse accezioni che i concetti di finzione e finzionalizzazione assumono nelle visioni prese in considerazione. Ma non solo. A differenziare le due prospettive, infatti, c'è soprattutto la diversa ambizione che le caratterizza. All’atteggiamento spregiudicato dei darwinisti letterari, pronti ad affermare l’origine evolutiva dello storytelling e quindi più tesi verso un’antropologia evoluzionistica, si contrappone il carattere strettamente culturale dell’elemento antropologico ricercato da Iser. In questo senso, pur nella consapevolezza della comune condivisione dell’ideale di un’antropologia della letteratura, si cercherà di mostrare, in seconda battuta, la radicale diversità in cui tale ideale si colloca. Così, pur nella convinzione che sia corretto vedere le tesi del darwinismo letterario come un ampiamento e un potenziamento di quelle iseriane – se si vuole una loro potenziale evoluzione – il presente contributo proverà a indicare alcuni limiti della posizione evoluzionista mostrando come, almeno per il momento, la bilancia della riflessione sulla narrazione penda decisamente a favore di una prospettiva antropologica più cautamente culturale

Rat Poison Kills a Pack of Eastern Coyotes, Canis latrans, in an Urban Area

We document the death of a pack of Eastern Coyotes (Canis latrans) from high levels of brodifacoum, a second generation poison that is the active ingredient in some forms of rat poison (e.g., d-Con®). The Coyotes died within a week of each other during late March/early April 2005. This incident indicates the vulnerability of wild animals to commercial over-the-counter rodenticides

p38 mitogen-activated protein kinase activation during platelet storage: Consequences for platelet recovery and hemostatic function in vivo

Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets

Mechanisms of Platelet Activation and Integrin αIIβ3

Platelet aggregation is not only an essential part of hemostasis, but also initiates acute coronary syndrome or ischemic stroke. The precise understanding of the activation mechanism of platelet aggregation is fundamental for the development of more effective agents against platelet aggregation. Adenosine diphosphate, thrombin, and thromboxane A2 activate platelet integrin αIIbβ3 through G protein-coupled receptors. G protein-mediated signaling pathways, which are initiated by Gq, G12/G13 or Gi, include phospholipase C with calcium signaling, Rho signaling, protein kinase C and phosphatidylinositol 3-kinase. Rap1b, Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I, Rap1-GTP-interacting adaptor molecule, and Akt are important proteins involved in G protein-mediated activation of integrin αIIbβ3. Binding of talin-1 and kindlin-3 to cytoplasmic domains of β3-integrin triggers a conformational change in the extracellular domains that increases its affinity for ligands, such as fibrinogen or von Willebrand factor. Fibrinogens act as bridges between adjacent platelets to generate a platelet aggregate

Kindlins, Integrin Activation and the Regulation of Talin Recruitment to αIIbβ3

Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment

The P2X1 receptor and platelet function

Extracellular nucleotides are ubiquitous signalling molecules, acting via the P2 class of surface receptors. Platelets express three P2 receptor subtypes, ADP-dependent P2Y1 and P2Y12 G-protein-coupled receptors and the ATP-gated P2X1 non-selective cation channel. Platelet P2X1 receptors can generate significant increases in intracellular Ca2+, leading to shape change, movement of secretory granules and low levels of αIIbβ3 integrin activation. P2X1 can also synergise with several other receptors to amplify signalling and functional events in the platelet. In particular, activation of P2X1 receptors by ATP released from dense granules amplifies the aggregation responses to low levels of the major agonists, collagen and thrombin. In vivo studies using transgenic murine models show that P2X1 receptors amplify localised thrombosis following damage of small arteries and arterioles and also contribute to thromboembolism induced by intravenous co-injection of collagen and adrenaline. In vitro, under flow conditions, P2X1 receptors contribute more to aggregate formation on collagen-coated surfaces as the shear rate is increased, which may explain their greater contribution to localised thrombosis in arterioles compared to venules within in vivo models. Since shear increases substantially near sites of stenosis, anti-P2X1 therapy represents a potential means of reducing thrombotic events at atherosclerotic plaques