Courses taught under Women\u27s Studies 1993 to present

List of course titles by semester from 1993 to 1998. 3 pages

Estudio citoquímico del glucogéno linfocitario en la leucemia linfática crónica pre y post-tratamiento con clorambucil

Fil: De Cosen, Rodica H.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Vesicle-Associated Membrane Protein 8 (VAMP8) is a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) selectively required for sequential granule-to-granule fusion

Compound exocytosis is found in many cell types and is the major form of regulated secretion in acinar and mast cells. Its key characteristic is the homotypic fusion of secretory granules. These then secrete their combined output through a single fusion pore to the outside. The control of compound exocytosis remains poorly understood. Although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as syntaxin 2, SNAP23 (synaptosome-associated protein of 23 kDa), and SNAP25 have been suggested to play a role, none has been proven. Vesicle-associated membrane protein 8 (VAMP8) is a SNARE first associated with endocytic processes but more recently has been suggested as an R-SNARE in regulated exocytosis. Secretion in acinar cells is reduced when VAMP8 function is inhibited and is less in VAMP8 knock-out mice. Based on electron microscopy experiments, it was suggested that VAMP8 may be involved in compound exocytosis. Here we have tested the hypothesis that VAMP8 controls homotypic granule-to-granule fusion during sequential compound exocytosis. We use a new assay to distinguish primary fusion events (fusion with the cell membrane) from secondary fusion events (granule-granule fusion). Our data show the pancreatic acinar cells from VAMP8 knock-out animals have a specific reduction in secondary granule fusion but that primary granule fusion is unaffected. Furthermore, immunoprecipitation experiments show syntaxin 2 association with VAMP2, whereas syntaxin 3 associates with VAMP8. Taken together our data indicate that granule-to-granule fusion is regulated by VAMP8 containing SNARE complexes distinct from those that regulate primary granule fusion

Influence of acute pancreatitis on the in vitro responsiveness of rat mesenteric and pulmonary arteries

<p>Abstract</p> <p>Background</p> <p>Acute pancreatitis is an inflammatory disease characterized by local tissue injury and systemic inflammatory response leading to massive nitric oxide (NO) production and haemodynamic disturbances. Therefore, the aim of this work was to evaluate the vascular reactivity of pulmonary and mesenteric artery rings from rats submitted to experimental pancreatitis.</p> <p>Male Wistar rats were divided into three groups: saline (SAL); tauracholate (TAU) and phospholipase A₂(PLA₂). Pancreatitis was induced by administration of TAU or PLA₂from <it>Naja mocambique mocambique </it>into the common bile duct of rats, and after 4 h of duct injection the animals were sacrificed. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP) and phenylephrine (PHE) in isolated mesenteric and pulmonary arteries were obtained. Potency (pEC₅₀) and maximal responses (E_MAX) were determined. Blood samples were collected for biochemical analysis.</p> <p>Results</p> <p>In mesenteric rings, the potency for ACh was significantly decreased from animals treated with TAU (about 4.2-fold) or PLA₂(about 6.9-fold) compared to saline group without changes in the maximal responses. Neither pEC₅₀nor E_MAXvalues for Ach were altered in pulmonary rings in any group. Similarly, the pEC₅₀and the E_MAXvalues for SNP were not changed in both preparations in any group. The potency for PHE was significantly decreased in rat mesenteric and pulmonary rings from TAU group compared to SAL group (about 2.2- and 2.69-fold, for mesenteric and pulmonary rings, respectively). No changes were seen in the E_MAXfor PHE. The nitrite/nitrate (NO_x^-) levels were markedly increased in animals submitted to acute pancreatitis as compared to SAL group, approximately 76 and 68% in TAU and PLA₂protocol, respectively.</p> <p>Conclusion</p> <p>Acute pancreatitis provoked deleterious effects in endothelium-dependent relaxing response for ACh in mesenteric rings that were strongly associated with high plasma NO_x^-levels as consequence of intense inflammatory responses. Furthermore, the subsensitivity of contractile response to PHE in both mesenteric and pulmonary rings might be due to the complications of this pathological condition in the early stage of pancreatitis.</p

The Absence of MIST1 Leads to Increased Ethanol Sensitivity and Decreased Activity of the Unfolded Protein Response in Mouse Pancreatic Acinar Cells

Background: Alcohol abuse is a leading cause of pancreatitis in humans. However, rodent models suggest that alcohol only sensitizes the pancreas to subsequent insult, indicating that additional factors play a role in alcohol-induced pancreatic injury. The goal of this study was to determine if an absence of MIST1, a transcription factor required for complete differentiation of pancreatic acinar cells in mice, increased the sensitivity to alcohol. Methods: Two to four month-old mice lacking MIST1 (Mist1 2/2) or congenic C57 Bl6 mice were placed on a Lieber-DeCarli diet (36 % of total kcal from ethanol and fat), a control liquid diet (36 % kcal from fat) or a regular breeding chow diet (22% kcal from fat). After six weeks, pancreatic morphology was assessed. Biochemical and immunofluorescent analysis was used to assess mediators of the unfolded protein response (UPR). Results: Ethanol-fed Mist1 2/2 mice developed periductal accumulations of inflammatory cells that did not appear in wild type or control-fed Mist1 2/2 mice. Wild type mice fed diets high in ethanol or fat showed enhancement of the UPR based on increased accumulation of peIF2a and spliced XBP1. These increases were not observed in Mist1 2/2 pancreatic tissue, which had elevated levels of UPR activity prior to diet exposure. Indeed, exposure to ethanol resulted in a reduction of UPR activity in Mist1 2/2 mice. Conclusions: Our findings suggest that an absence of MIST1 increases the sensitivity to ethanol that correlated wit

Distinct phosphorylation requirements regulate cortactin activation by TirEPEC and its binding to N-WASP

<p>Abstract</p> <p>Background</p> <p>Cortactin activates the actin-related 2/3 (Arp2/3) complex promoting actin polymerization to remodel cell architecture in multiple processes (e.g. cell migration, membrane trafficking, invadopodia formation etc.). Moreover, it was called the Achilles' heel of the actin cytoskeleton because many pathogens hijack signals that converge on this oncogenic scaffolding protein. Cortactin is able to modulate N-WASP activation <it>in vitro </it>in a phosphorylation-dependent fashion. Thus Erk-phosphorylated cortactin is efficient in activating N-WASP through its SH3 domain, while Src-phosphorylated cortactin is not. This could represent a switch on/off mechanism controlling the coordinated action of both nucleator promoting factors (NPFs). Pedestal formation by enteropathogenic <it>Escherichia coli </it>(EPEC) requires N-WASP activation. N-WASP is recruited by the cell adapter Nck which binds a major tyrosine-phosphorylated site of a bacterial injected effector, Tir (translocated intimin receptor). Tir-Nck-N-WASP axis defines the current major pathway to actin polymerization on pedestals. In addition, it was recently reported that EPEC induces tyrosine phosphorylation of cortactin.</p> <p>Results</p> <p>Here we demonstrate that cortactin phosphorylation is absent on N-WASP deficient cells, but is recovered by re-expression of N-WASP. We used purified recombinant cortactin and Tir proteins to demonstrate a direct interaction of both that promoted Arp2/3 complex-mediated actin polymerization <it>in vitro</it>, independently of cortactin phosphorylation.</p> <p>Conclusion</p> <p>We propose that cortactin binds Tir through its N-terminal part in a tyrosine and serine phosphorylation independent manner while SH3 domain binding and activation of N-WASP is regulated by tyrosine and serine mediated phosphorylation of cortactin. Therefore cortactin could act on Tir-Nck-N-WASP pathway and control a possible cycling activity of N-WASP underlying pedestal formation.</p

Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo

Cortactin is required for endothelial barrier function and leukocyte recruitment in vivo

Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia—membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin—another prominent Arp2/3 complex regulator—and ADF/cofilin—previously implicated in driving both filament nucleation and disassembly—were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh

The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

Background: Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined. Methodology/Principal Findings: In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation. Conclusion: Our results suggest a novel function of phosphorylation-dependent cortactin signaling downstream fro

A Novel Pseudopodial Component of the Dendritic Cell Anti-Fungal Response: The Fungipod

Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the “fungipod”, formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7±5.6 µm long and 1.8±0.67 µm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225±55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 µm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion