A logical model provides insights into T cell receptor signaling

Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.     

Population structure,life cycle,and trophic niche of the glacial relict amphipod,Gammaracanthus lacustris,in a large boreal lake

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Paleoenvironmental analysis

New analysis has been carried out concerning the palaeoenvironmental reconstruction of some Italian sites dating from the Middle Pleistocene to the Bronze Age. Different aspects have been investigated on each site considering the data collected. The following sites have been analyzed: Isernia La Pineta (Molise); Visogliano and Caverna degli Orsi (Tieste); Toirano Caves (Liguria); Grotta Paglicci (Gargano); Riparo del Molare (Salerno); Grotta del Cavallo (Lecce); Castellaro Lagusello (Monzambano, Mantova).     

Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.     

Characterization and functional identification of a novel plant 4,5-extradiol dioxygenase involved in betalain pigment biosynthesis in Portulaca grandiflora

Betalains are pigments that replace anthocyanins in the majority of families of the plant order Caryophyllales. Betalamic acid is the common chromophore of betalains. The key enzyme of the betalain biosynthetic pathway is an extradiol dioxygenase that opens the cyclic ring of dihydroxy-phenylalanine (DOPA) between carbons 4 and 5, thus producing an unstable seco-DOPA that rearranges nonenzymatically to betalamic acid. A gene for a 4,5-DOPA-dioxygenase has already been isolated from the fungus Amanita muscaria, but no homolog was ever found in plants. To identify the plant gene, we constructed subtractive libraries between different colored phenotypes of isogenic lines of Portulaca grandiflora (Portulacaceae) and between different stages of flower bud formation. Using in silico analysis of differentially expressed cDNAs, we identified a candidate showing strong homology at the level of translated protein with the LigB domain present in several bacterial extradiol 4,5-dioxygenases. The gene was expressed only in colored flower petals. The function of this gene in the betalain biosynthetic pathway was confirmed by biolistic genetic complementation in white petals of P. grandiflora genotypes lacking the gene for color formation. This gene named DODA is the first characterized member of a novel family of plant dioxygenases phylogenetically distinct from Amanita sp. DOPA-dioxygenase. Homologs of DODA are present not only in betalain-producing plants but also, albeit with some changes near the catalytic site, in other angiosperms and in the bryophyte Physcomitrella patens. These homologs are part of a novel conserved plant gene family probably involved in aromatic compound metabolism.     

Cyanophage diversity, inferred from g20 gene analyses, in the largest natural lake in France, Lake Bourget

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.     

Changes of glial-neuronal interaction and metabolism after a subconvulsive dose of pentylenetetrazole

Pentylenetetrazole (PTZ) injection causes seizures in rodents and this is used in several models of epilepsy. In the present study a low dose (20 mg/kg) was injected into rats in order to analyze metabolic disturbances caused by subconvulsive amounts of PTZ. Intraperitoneal injection of PTZ was followed, 30 min later, by injection of [1-(13C)]glucose plus [1,2-(13C)]acetate and 15 min thereafter decapitation. Analyses of extracts from cerebrum, subcortex and cerebellum were performed using 13C NMRS and HPLC. Whereas convulsive doses of PTZ lead to most pronounced changes in cerebellum [J. Neurochem. 85 (2003) 1200], it could be shown that subconvulsive doses affected mainly amino acid metabolism in cerebrum. In glutamatergic neurons in the cerebrum PTZ affected both the metabolic and releasable pools of glutamate, whereas, in the subcortex and cerebellum only the metabolic pool was affected. This could be deducted from the findings that less [4-(13C)]glutamine, [3,4-(13C)]glutamate and [2-(13C)]aspartate, which are labeled from [1-(13C)]glucose, were detected in this area. Glial metabolism was also changed as evidenced by the decreased pyruvate carboxylation versus pyruvate dehydrogenation ratio both in cerebrum and subcortex. Comparison between convulsive and nonconvulsive doses of PTZ lead to the hypothesis that changes observed in the cerebellum are mainly due to seizures, whereas those in cerebrum and subcortex are coupled to the action of the chemical stimulant.