Cooperative Vehicular Traffic Monitoring in Realistic Low Penetration Scenarios: The COLOMBO Experience.

The relevance of effective and efficient solutions for vehicle traffic surveillance is widely recognized in order to enable advanced strategies for traffic management, e.g., based on dynamically adaptive and decentralized traffic light management. However, most related solutions in the literature, based on the powerful enabler of cooperative vehicular communications, assume the complete penetration rate of connectivity/communication technologies (and willingness to participate in the collaborative surveillance service) over the targeted vehicle population, thus making them not applicable nowadays. The paper originally proposes an innovative solution for cooperative traffic surveillance based on vehicular communications capable of: (i) working with low penetration rates of the proposed technology and (ii) of collecting a large set of monitoring data about vehicle mobility in targeted areas of interest. The paper presents insights and lessons learnt from the design and implementation work of the proposed solution. Moreover, it reports extensive performance evaluation results collected on realistic simulation scenarios based on the usage of iTETRIS with real traces of vehicular traffic of the city of Bologna. The reported results show the capability of our proposal to consistently estimate the real vehicular traffic even with low penetration rates of our solution (only 10%).

In vitro characterization of cholinesterases in the earthworm Eisenia andrei.

Assessment of pollution impact in soil ecosystems has become a priority and interest has grown concerning the use of invertebrates as sentinel organisms. Inhibition of cholinesterase (ChE) activity has a great potential as a biomarker of pesticide exposure, and we evaluated the ChE kinetic parameters in the earthworm Eisenia andrei in the presence of acetylthiocholine (ASCh), proprionylthiocholine (PSCh) and butyrylthiocholine (BSCh). The highest ChE activity was found in the presence of ASCh and PSCh (42.45 and 49.82 nmol min(-1) mg protein(-1), respectively). BSCh was hydrolyzed at a rate of 4.04 nmol min(-1) mg protein(-1), but the time course did not reach a plateau under our experimental conditions. Km values were 0.142+/-0.006 and 0.183+/-0.053 mM for ASCh and PSCh, respectively. ASCh and PSCh hydrolysis were significantly inhibited by eserine (IC50 values were 1.44 x 10(-8) and 1.20 x 10(-8) M, respectively) and by carbaryl (IC50 values of 5.75 x 10(-9) and 4.79 x 10(-9) M). The presence of different ChEs in tissues from E. andrei was assessed by using selective inhibitors for AChE (BW284c51) and BChE (iso-OMPA). BW284c51 strongly reduced ASCh and PSCh hydrolysis and slightly affected that of BSCh, while iso-OMPA was without effect in all cases.

Modulation of adenyl cyclase activity in the gills of Tapes philippinarum.

Adenyl cyclase (AC) plays a pivotal role in cell signaling. The AC system of bivalves has received little attention so far, and our study has been addressed to the characterization of AC properties in the gills of T. philippinarum. The enzyme showed a Km value of 0.77 mM for ATP in the presence of 5 mM Mg2+; in the absence of agonists, it was poorly affected by GTP, while it was stimulated by GTPgammaS and GppNHp up to 14-fold and 4-fold, respectively. Similarly to other invertebrates, the enzyme activity was scarcely stimulated by forskolin. The receptor agonist serotonin (5-HT) significantly stimulated the AC activity, and the pharmacological profile of the 5-HT receptor/s was as follows: (+)butaclamol > dihydroergocryptine > methysergide > prazosin > yohimbine. The AC activity was assessed in vitro in the presence of tributyltin chloride and HgCl2, which reduced the AC activity only at the highest dose tested (10-100 microM). Our data indicate the presence of a membrane-bound AC in gill membranes of T. philippinarum, coupled to Gs proteins and to a specific class of 5-HT receptors. Such receptors show a pharmacological profile slightly different from that reported for 5-HT invertebrate receptors cloned so far.

Cd2+ and Hg2+ affect glucose release and cAMP-dependent transduction pathway in isolated eel hepatocytes.

Isolated hepatocytes of the European eel (Anguilla anguilla) have been used as experimental model to characterize the effects of Cd(2+) and Hg(2+) on either basal or epinephrine-stimulated glucose release. Cd(2+) strongly reduced glucose output from cells perifused in BioGel P4 columns and challenged with epinephrine, with a maximum inhibition of 95% reached at 10 microM (IC(50) 0.04 microM). The epinephrine-stimulated glucose output was also reduced by Hg(2+), although a significant inhibition of about 60% was achieved only at 10 microM (IC(50) 5 microM). The possible influence of Cd(2+) and Hg(2+) on adenylyl cyclase/cAMP transduction pathway has been investigated, since this system is known to play a pivotal role in the regulation of fish liver glycogen breakdown and consequent glucose release. Micromolar concentrations of both heavy metals significantly reduced the epinephrine-modulated cAMP levels in isolated eel hepatocytes, in good agreement with the reduction of glucose output. Cd(2+) and Hg(2+) also significantly reduced basal and epinephrine-stimulated adenylyl cyclase activity in liver membrane preparations. A competitive inhibition with respect to Mg(2+) was shown by Cd(2+) and Hg(2+), which significantly reduced the affinity of the allosteric activator for the adenylyl cyclase system. Apparent Km for Mg(2+) was 4.35 mM in basal conditions, and increased to 9.1 and 7.1 mM in the presence of 10 microM Cd(2+) and Hg(2+), respectively. These results indicate that Cd(2+) and Hg(2+) may impair a crucial intracellular transduction pathway involved in the adrenergic control of glucose metabolism, but also in several other routes of hormonal regulation of liver functions.

Hsp70 expression in thermally stressed Ostrea edulis, a commercially important oyster in Europe.

Synthesis of heat shock proteins (Hsps) in response to elevated temperatures and other denaturing agents is a common feature of prokaryotic and eukaryotic cells. The heat-induced expression of Hsp70 family members in the gills and mantle of Ostrea edulis, a highly valued fisheries resource inhabiting primarily estuarine environments, has been studied. O edulis is exposed to a variety of natural and anthropogenic stresses in the environment. Two isoforms of about 72 kDa and 77 kDa were constitutively present in unstressed organisms, reflecting the housekeeping function performed by these proteins under normal circumstances. Their expression in animals undergoing thermal stress was highly variable, and on the average, little change occurred under different experimental conditions. A third isoform of about 69 kDa was induced in both tissues after exposure to > or = 32 degrees C; its synthesis was detected within 4 hours of poststress recovery at 15 degrees C, reaching the maximum expression after 24 hours in the gills and after 48 hours in the mantle and declining thereafter. Hsp69 expression was low at 38 degrees C, a temperature lethal for about 50% of the individuals tested. Densitometric analysis of Western blots revealed that Hsp69 was mostly responsible for the significant heat-induced overexpression of Hsp70s in O edulis. Comparison with heat shock responses in tissues of Crassostrea gigas indicated a similar pattern of Hsp70 expression. In this organism, however, Hsp69 was induced after exposure to > or = 38 degrees C. We conclude that tissue expression of Hsp69 in O edulis, and possibly other bivalves, is an early sign of thermal stress; determining whether these changes also correlate with other major environmental stresses is the goal of ongoing studies.

Identification and properties of a Gs protein in catfish liver membranes.

The presence of G proteins and their involvement in adrenergic signaling has been investigated in catfish (Ictalurus melas) liver membranes. Adenylyl cyclase activity was potently stimulated by the nonhydrolyzable analog of GTP, [35S]guanosine 5'-O-(gamma-thiotriphosphate) (GTPgammaS) (maximal activation of about eightfold at 10(-5) M; half-maximal activation at 1.31 x 10(-7) M), and reduced by the competitive inhibitor of GTP, GDPbetaS (70% maximal inhibition at 10(-4) M; half-maximal inhibition at 1.98 x 10(-7) M). Forskolin dramatically enhanced enzyme activity (up to about 3500% at 100 microM), and its action was not affected by guanine nucleotides, confirming that the diterpene effect occurred only at targets downstream of the G proteins. Receptor-dependent G protein activity was evaluated by a [(35)S]GTPgammaS binding assay. At 100 microM GDP, 100 mM NaCl, and 5 mM MgCl2, after an incubation of 90 min at 20 degrees, a Kd of 18.6 nM and a Bmax of 105.7 pmol/mg protein for [35S]GTPgammaS binding to catfish liver membranes were determined. The binding of the tracer was enhanced by 1 microM epinephrine, up to a maximum of 158%, and inhibited by NF 449, a G(s)alpha-selective antagonist with half-maximal effect in the micromolar range. Immunoblotting analysis with a specific anti-G(s)alpha antibody revealed a single band of about 45 kDa mass. This result represents the first demonstration of the presence of G protein alpha(s) subunits in the liver of an ectothermal vertebrate.

G proteins immunodetection and adrenergic transduction pathways in the liver of Anguilla anguilla.

G proteins are members of a highly conserved superfamily of GTPases, which includes heterotrimeric (alpha, beta, gamma) proteins acting as critical control points for transmembrane signaling. In ectothermal vertebrates, knowledge about these proteins is scarce, and our work provides the first demonstration that G(s), G(q), and G(i) proteins are all present in the liver of a fish. G(q)alpha subunits of about 42 kDa have been identified in European eel (Anguilla anguilla) liver membranes, supporting previous reports about the existence of hormone transduction pathways coupled to inositol 1,4,5-trisphosphate/Ca(2+) enhancement in fish hepatocytes. Although two G(s)alpha proteins of about 45 and 52 kDa have been reported in mammals, a single isoform of approximately 45 kDa has been recognized in eel liver. G(s)alpha and G(q)alpha proteins are involved in the epinephrine transduction pathway, leading to cAMP and Ca(2+) intracellular increments, respectively. Interestingly, both messengers significantly stimulated glucose release from eel hepatocytes but with a different time course. In fact, the Ca(2+)-dependent glucose output preceded the cAMP-mediated release by about 7 min. G(i)alpha subunits of about 40 kDa were also immunodetected, suggesting the presence of hormone receptors leading to adenylyl cyclase inhibition in eel liver; however, alpha(2)- adrenoreceptor ligands were ineffective on both enzyme activity and glucose release.