GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.     

Physiological and theoretical analysis of K+ currents controlling discharge in neonatal rat mesencephalic trigeminal neurons

Whole cell voltage- and current-clamp recordings were obtained from mesencephalic trigeminal sensory (Mes 5) neurons identified visually in thin brain stem slices of neonatal rats with the use of infrared video microscopy. These cells exhibited accommodation in spike discharge responses to depolarizing current injection protocols whose duration differed as a function of holding potential (-50 vs. -65 mV). Several spikes were elicited before the membrane response accommodated from -50 mV, whereas from -65 mV only single action potentials were evoked. In response to similar protocols, application of the K+ channel blocker 4-aminopyridine (4-AP) (50 microM to 2 mM) caused sustained repetitive spiking whereas tetraethylammonium (TEA) (10-30 mM) did not cause repetitive spiking. In voltage clamp, 4-AP application (100 microM) revealed a sustained outward current (I4-AP) that was active between -60 and -30 mV. I4-AP was responsible for suppressing sustained repetitive spiking behavior, producing accommodation under normal circumstances. TEA application in voltage clamp revealed a sustained outward current evoked positive to -40 mV. Two transient outward currents (TOCs) were identified by prepulse protocols typically used to characterize A-type currents: a 4-AP-insensitive fast TOC, and a slow TOC (ITOC-S) sensitive to 4-AP (> 500 microM). A Ca(2+)-dependent outward current that activated positive to -30 mV was also characterized. A mathematical model of a Mes 5 neuron was assembled from our voltage-clamp records to simulate the dynamic interaction of outward currents during membrane excitation. We conclude that in Mes 5 neurons, the 4-AP-sensitive currents ITOC-S and I4-AP determine the duration of spike trains. In particular, the noninactivating I4-AP determines whether cells exhibit sustained repetitive discharge or accommodate in response to depolarizing current. Neurotransmitter modulation of this current or modulation of the resting membrane potential could modify the output properties of Mes 5 neurons, and therefore the properties of these currents must be incorporated into our current understanding of how these cells contribute to shaping oral-motor pattern generation.     

Autosensitized Photooxidation of the Rose Hip (Wild Rose) Oil - Mechanism and Quantum Yield for Singlet Molecular Oxygen O2 (1 Δg) Generation

The autosensitized photooxidation of the fatty acid components of rose hip (Wild Rose) oil was studied. Two important facts arise from the present investigation: a) Fatty acids of rose hip oil decompose upon visible light irradiation in the absence of external sensitizers by a mechanism that involves O₂(¹Δ_g). The pigments present in rose hip oil themselves efficiently sensitize the generation of O₂(¹Δ_g) with a quantum yield of 0.038 (upper limit). b) Visible irradiation also produces the degradation of the anthocyanines, native pigments which could behave as O₂(¹Δ_g) sensitizers in the oil. In this case a mechanism involving the production of superoxide ions by the pigment and further attack of the radicals on the phenol groups of the anthocyanine could account for the observed experimental evidences. These results should be carefully considered in a practical sense when rose hip oil is stored or employed under daylight conditions. Exposure to visible radiation may cause irreversible transformations in rose hip oil.     

Identifying a putative common binding site shared by substance P receptor and an anti-substance P monoclonal antibody

Substance P G-protein coupled receptor and the antigen recognitionsite of a monoclonal antibody raised against substance P sharea stretch of five contiguous identical amino acids. This observationprompted us to build an atomic model of both the receptor andthe antibody and to analyse their common features. In particular,we report here that a pocket of similar size and compositionis present in both proteins, strongly suggesting a similarityin the mode of binding of both macromolecules to substance P.From the analysis of our models, the available data on the modeof binding of the antibody to substance P and recent data onsubstance P receptor mutants, we concluded that the pocket isvery likely to be involved in binding of the C-terminal 'messagesequence' of the tachykinin. This allowed us to suggest specificsite-directed mutants of the receptor which should shed somelight on the mechanism of peptide recognition by G-protein coupledreceptors.     

Sulfate attack of concrete building foundations induced by sewage waters

A case history of a severe degradation of concrete foundation plinths and piers of an about-35-year-old building located in Northern Italy is described. Significant amounts of gypsum, near ettringite and/or thaumasite were detected by X-ray diffraction analyses performed on ground concrete samples. Large gypsum crystals were mainly located at the interface between the cement paste and aggregates, as observed by scanning electron microscopy coupled to energy-dispersive spectroscopy microanalysis. The degradation effects increased with decreasing the distance of concrete structures from an absorbing well located in the courtyard of the building. The well was recognized as the sulfate source due to the microorganism metabolism of sulfur compounds present in the sewage. Consequences of this attack were a very poor bond strength between cement paste and aggregates and a severe cracking of the concrete cover of the steel reinforcement.     

Sintering behaviour of gel-derived powders

85Al₂O₃-15ZrO₂ (wt%) powders were synthesized by gel precipitation starting from AlCl₃ · 6H₂O and ZrCl₄ solutions and dried by two different methods: (i) by spray-drying and (ii) via a sol-gel route by n-octanol in a pilot plant. The particles by process (i) were spherical granules of diameter 15 m, and those by process (ii) were microspheres of 25 m. The powders were characterized in terms of morphology, particle size distribution, surface area, weight loss and crystallization behaviour at different temperatures. Shrinkage, microstructure, density and pore size distribution were evaluated on compacts at different temperatures to study the sintering kinetics. Experimental observations suggest that (a) the use of controlled-geometry powders allows one to obtain high green densities; and (b) the sintering of gel-derived powders develops in two steps: during the first one, sintering mainly takes place inside each microsphere (or granule), and during the second step, mainly between the microspheres (or granules). At temperatures > 1100°C, sintering produces shrinkage of the microspheres (or granules) leading to pore formation between them, which prevents the achievement of high densities. Only by using hot pressing is it possible to obtain theoretical densities and high mechanical properties.     

Degree-Optimal Routing for P2P Systems

We define a family of Distributed Hash Table systems whose aim is to combine the routing efficiency of randomized networks—e.g. optimal average path length O(log ² n/δlog δ) with δ degree—with the programmability and startup efficiency of a uniform overlay—that is, a deterministic system in which the overlay network is transitive and greedy routing is optimal. It is known that Ω(log n) is a lower bound on the average path length for uniform overlays with O(log n) degree (Xu et al., IEEE J. Sel. Areas Commun. 22(1), 151–163, 2004). Our work is inspired by neighbor-of-neighbor (NoN) routing, a recently introduced variation of greedy routing that allows us to achieve optimal average path length in randomized networks. The advantage of our proposal is that of allowing the NoN technique to be implemented without adding any overhead to the corresponding deterministic network. We propose a family of networks parameterized with a positive integer c which measures the amount of randomness that is used. By varying the value c, the system goes from the deterministic case (c=1) to an “almost uniform” system. Increasing c to relatively low values allows for routing with asymptotically optimal average path length while retaining most of the advantages of a uniform system, such as easy programmability and quick bootstrap of the nodes entering the system. We also provide a matching lower bound for the average path length of the routing schemes for any c. This work was partially supported by the Italian FIRB project “WEB-MINDS” (Wide-scalE, Broadband MIddleware for Network Distributed Services), .     

Cell-protection mechanism through autophagy in HGFs/<Emphasis Type="Italic">S. mitis</Emphasis> co-culture treated with Chitlac-nAg

Silver-based products have been proven to be effective in retarding and preventing bacterial growth since ancient times. In the field of restorative dentistry, the use of silver ions/nanoparticles has been explored to counteract bacterial infections, as silver can destroy bacterial cell walls by reacting with membrane proteins. However, it is also cytotoxic towards eukaryotic cells, which are capable of internalizing nanoparticles. In this work, we investigated the biological effects of Chitlac-nAg, a colloidal system based on a modified chitosan (Chitlac), administered for 24–48?h to a co-culture of primary human gingival fibroblasts and Streptococcus mitis in the presence of saliva, developed to mimic the microenvironment of the oral cavity. We sought to determine its efficiency to combat oral hygiene-related diseases without affecting eukaryotic cells. Cytotoxicity, reactive oxygen species production, apoptosis induction, nanoparticles uptake, and lysosome and autophagosome metabolism were evaluated. In vitro results show that Chitlac-nAg does not exert cytotoxic effects on human gingival fibroblasts, which seem to survive through a homoeostasis mechanism involving autophagy. That suggests that the novel biomaterial Chitlac-nAg could be a promising tool in the field of dentistry.