Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.     

C. elegans Eats Its Own Intestine to Make Yolk Leading to Multiple Senescent Pathologies

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Determination of the optimal therapeutic protocols in cancer immunotherapy

Cancer immunotherapy aims at eliciting an immune system response against the tumor. However, it is often characterized by toxic side-effects. Limiting the tumor growth and, concurrently, avoiding the toxicity of a drug, is the problem of protocol design. We formulate this question as an optimization problem and derive an algorithm for its solution. Unlike the standard optimal control approach, the algorithm simulates impulse-like drug administrations. It relies on an exact computation of the gradient of the cost function with respect to any protocol by means of the variational equations, that can be solved in parallel with the system. In comparison with previous versions of this method [F. Castiglione, B. Piccoli, Optimal control in a model of dendritic cell transfection cancer immunotherapy, Bull. Math. Biol. 68 (2006) 255-274; B. Piccoli, F. Castiglione, Optimal vaccine scheduling in cancer immunotherapy, Physica A. 370 (2) (2007) 672-680], we optimize both the timing and the dosage of each administration and introduce a penalty term to avoid clustering of subsequent injections, a requirement consistent with the clinical practice. In addition, we implement the optimization scheme to simulate the case of multi-therapies. The procedure works for any ODE system describing the pharmacokinetics and pharmacodynamics of an arbitrary number of therapeutic agents. In this work, it was tested for a well known model of the tumor-immune system interaction [D. Kirschner, J.C. Panetta, Modeling immunotherapy of tumor-immune interaction, J. Math. Biol. 37 (1998) 235-252]. Exploring three immunotherapeutic scenarios (CTL therapy, IL-2 therapy and combined therapy), we display the stability and efficacy of the optimization method, obtaining protocols that are successful compromises between various clinical requirements.     

Study of the surface morphology of a cholesteryl tethering system for lipidic bilayers

The immobilization of functional molecules embedded in lipidic membranes onto inorganic substrates is of great interest for numerous applications in the fields of biosensors and biomaterials. We report on the preparation and the morphological characterization of a tethering system for lipidic bilayers, which is based on cholesteryl derivatives deposited on hydrophilic surfaces by self-assembling and microcontact printing techniques. The investigation of the structural properties of the realized films by atomic, lateral, and surface potential microscopy allowed us to assess the high quality of the realized cholesteryl layers.     

Mitochondrial pH monitored by a new engineered green fluorescent protein mutant

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK(a) (pK(a)') around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca(2+) ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca(2+) accumulation by this organelle. Finally, we demonstrate that Ca(2+) mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.     

TRAIL-related neurotoxicity implies interaction with the Wnt pathway in human neuronal cells in vitro

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is involved in amyloid beta dependent neurotoxicity via the extrinsic pathway. Recently, several genes modulating TRAIL cytotoxicity have been characterized, providing evidence for a role of wingless-type mouse mammary tumor virus integration site family (Wnt), Jun-N-terminal kinase and other pathways in increased cell susceptibility to the cytokine. We investigated whether neurotoxic effects of TRAIL could be due to modulation of the Wnt signaling pathway. Western blot analysis of Wnt in SH-SY5Y human neuroblastoma cells showed significantly decreased Wnt expression in cultures treated with TRAIL. Correspondingly, both phosphorylation of glycogen synthase kinase 3 beta and degradation of cytoplasmic β-catenin were increased, as well as phosphorylation of the τ protein, bringing about the picture of neuronal damage. As a counterproof of the interaction of TRAIL with the Wnt pathway, the addition of the specific glycogen synthase kinase 3 beta inhibitor SB216763 resulted in rescue of a significant percent of cells from TRAIL-induced apoptosis. The rescue was total when the caspase 8 inhibitor z-IETD-FMK was added in combination with SB216763. Results show that, probably, in addition to triggering caspase signaling, TRAIL also interferes with the Wnt pathway, additionally concurring to neuronal damage. These data suggest that the Wnt pathway substantially contributes to the TRAIL-related neurotoxicity and indicate the TRAIL system as a candidate target for pharmacological treatment of Alzheimer's disease and related disorders.