Miguel Angel Sanjuán

T Cell Activation In Vivo Targets Diacylglycerol Kinase α to the Membrane: A Novel Mechanism for Ras Attenuation

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGKα, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGKα, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGKα to the membrane fraction. At early time points, DGKα translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGKα activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGKα mutants. We show that membrane localization of DGKα is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGKα, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.

Type Iα phosphatidylinositol 4-phosphate 5-kinase is a putative target for increased intracellular phosphatidic acid

Despite the fact that phosphatidic acid (PtdOH) has been implicated as a lipid second messenger for nearly a decade, its intracellular targets have remained unclear. We sought to investigate how an increase in the level of PtdOH could modulate phosphatidylinositol 4‐phosphate 5‐kinase (PIPkin), an enzyme involved in phosphatidylinositol 4,5‐bisphosphate synthesis. Transfection of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)‐tagged type Iα PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transfected PAE cells were stimulated with lyso‐PtdOH, increased PIPkin activity was found to be associated with HA immunoprecipitates in an in vitro assay. This PIPkin activation was found to be greatly reduced by prior treatment of the cells with 1‐butanol, thereby implicating phospholipase D (PLD) as the in vivo generator of PtdOH. In order to determine if the PtdOH‐dependent activation of type Iα PIPkin was dictated by a specific molecular composition of PtdOH, the wild type murine and porcine α isoforms of diacylglycerol kinase (DGK) were individually co‐transfected along with type Iα PIPkin. Under these conditions an increase in type Iα PIPkin lipid kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type Iα PIPkin was co‐transfected with either the human DGKϵ isoform or a kinase‐dead mutant of the murine DGKα isoform. These results provide the first direct evidence for the unification of the production of saturated/monounsaturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type Iα PIPkin by this lipid second messenger.

Expression of a catalytically inactive form of diacylglycerol kinase α induces sustained signaling through RasGRP

Regulating the generation and clearance of lipid second messengers, such as diacylglycerol (DAG), is critical for the correct propagation of intracellular signaling pathways. DAGK type α acts as a negative modulator of the DAG levels generated during T cell activation, which is initiated by triggering of the endogenous T cell receptor (TCR), as well as by stimulation of an ectopically expressed human muscarinic type 1 receptor. Here we show that stimulation of either of these receptors causes rapid, transient membrane translocation of the recently discovered Ras guanyl nucleotide release protein (RasGRP), followed by activation of mitogen‐activated protein kinase (MAPK). When cells expressing a catalytically inactive form of DAGKα were analyzed, however, similar agonist stimulation resulted in sustained signaling through RasGRP and MAPK. Biochemical analysis showed that expression of kinase‐dead diacylglycerol kinase α (DGKα) led to a greater, more sustained, DAG accumulation following receptor stimulation. These results suggest that, in T cells, agonist‐stimulated DAG generation is the key factor controlling activation of the Ras/MAPK signaling pathway through membrane translocation of RasGRP. Moreover, we demonstrate that through the modulation of the intracellular level of agonist‐stimulated DAG, DGKα alters Ras activation and downstream signaling dramatically, a process of utmost importance for sound immunological function.

Role of the Diacylglycerol Kinase α-Conserved Domains in Membrane Targeting in Intact T Cells

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKα is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKα domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr335. This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

Carbonation resistance of one industrial mortar used as a concrete coating

Abstract In aggressive environments, concrete itself may not be enough to protect the reinforcement against carbon dioxide penetration. This gas reacts with the portlandite of the concrete to form calcium carbonate. This process leads to a pH reduction and, therefore, promotes the depassivation of the steel reinforcement in reinforced concretes. Therefore, a supplementary protection method such as coating with a mortar, as carbonation barrier can be used to provide adequate durability. Experimental data are presented to illustrate the effect of three different testing levels of CO 2 on an industrial mortar applied to a concrete base in order to evaluate its performance as an anti-carbonation barrier. The results from the coated concrete are compared with the carbonation resistance of the uncoated plain concrete. The carbonation depth was determined using a phenolphthalein pH-indicator. A clear reduction in carbonation was observed when the mortar was applied. The validity of the accelerated testing method, which consists of placing the mortar in a high carbon dioxide concentration chamber for a controlled time, and carbonation coefficients to assess coating effectiveness are discussed. The use of 100% carbon dioxide is highly questionable for accelerated carbonation testing.

Interleukin-2 Causes an Increase in Saturated/Monounsaturated Phosphatidic Acid Derived from 1,2-Diacylglycerol and 1-O-Alkyl-2-acylglycerol

Phosphatidic acid generation through activation of diacylglycerol kinase α has been implicated in interleukin-2-dependent T-lymphocyte proliferation. To investigate this lipid signaling in more detail, we characterized the molecular structures of the diradylglycerols and phosphatidic acids in the murine CTLL-2 T-cell line under both basal and stimulated conditions. In resting cells, 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol subtypes represented 44 and 55% of total diradylglycerol, respectively, and both showed a highly saturated profile containing primarily 16:0 and 18:1 fatty acids. 1-O-Alk-1′-enyl-2-acylglycerol represented 1–2% of total diradylglycerol. Interleukin-2 stimulation did not alter the molecular species profiles, however, it did selectively reduce total 1-O-alkyl-2-acylglycerol by over 50% at 15 min while only causing a 10% drop in 1,2-diacylglycerol. When radiolabeled CTLL-2 cells were challenged with interleukin-2, no change in the cellular content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of diradylglycerol. In addition, interleukin-2 failed to stimulatede novo synthesis of diradylglycerol. Structural analysis revealed approximately equal amounts of 1,2-diacyl phosphatidic acid and 1-O-alkyl-2-acyl phosphatidic acid under resting conditions, both containing only saturated and monounsaturated fatty acids. After acute (2 and 15 min) interleukin-2 stimulation the total phosphatidic acid mass increased, almost entirely through the formation of 1-O-alkyl-2-acyl species. In vitro assays revealed that both 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol were substrates for 1,2-diacylglycerol kinase α, the major isoform in CTLL-2 cells, and that the lipid kinase activity was almost totally inhibited by R59949. In conclusion, this investigation shows that, in CTLL-2 cells, 1,2-diacylglycerol kinase α specifically phosphorylates a pre-existing pool of 1-O-alkyl-2-acylglycerol to form the intracellular messenger 1-O-alkyl-2-acyl phosphatidic acid.

Polypropylene-fibre-reinforced mortar mixes: Optimization to control plastic shrinkage

Volume changes which occur as a newly cast mortar is exposed to drying conditions will result in tensile stresses and lead to cracking of the mortar and concrete at early ages. Because of the growing interest in predicting the tensile shrinkage stresses that give rise to cracking in concrete, and the increasing use of polypropylene-fibre-reinforced cementitious materials, this research has been carried out to investigate how mix proportions influence the plastic shrinkage of polypropylene-fibre-reinforced mortars. This paper describes an experimental approach to the problem, which was intended to investigate the optimization of polypropylene-fibre-reinforced mortar mixes in order to minimize plastic shrinkage. The method selected is based on factorial design of experiments in which the parameters considered are the cement/sand and water/cement ratios and the polypropylene fibre content. The test results indicate that polypropylene fibres added to mortars reduce the plastic shrinkage. As a first result, a mathematical equation which allows prediction of the plastic shrinkage as a function of the mix design is given.

A testing method for measuring plastic shrinkage in polypropylene fibre reinforced mortars

The use of concretes reinforced with a low volume of polypropylene fibres added as secondary reinforcement, mainly to control cracking due to plastic shrinkage at early stages is of recent interest. In this paper, a method for measuring plastic shrinkage of mortars is proposed. The influence of the water/cement ratio, cement/sand ratio and amount of polypropylene fibres on the plastic shrinkage of mortars is also studied. The method presented in this paper gives information about the quality of the material studied in terms of plastic shrinkage and potential cracking.

Influence of the age on air permeability of concrete

A great interest is being taken in the measurement of concrete permeability because of increased concerns about the durability of concrete when exposed to aggressive environments. However, the variation of the concrete permeability with time has not been systematically studied. Air permeability results recorded for 20 years in six 0.2 m3 concrete slabs are presented. These results have a qualitative value as boundary effects are unknown. The great difference in flow between the specimens with the same mix design, casting and curing procedure, which have been kept in the same testing room sheltered from rain, is noticeable. The air permeability coefficient increases with time, reaching an almost stable value after 20 years. The pore evaporated water creates a path for air flow through the concrete and this is supposed to be the main cause of the increase in permeability. The method presented in this paper gives reliable information about the quality of the concrete studied in terms of air permeability and hence, durability.

Model for predicting plastic shrinkage of polypropylene reinforced mortars

There is increasing interest in the use of concretes reinforced with a low volume of polypropylene fibres added as secondary reinforcement, mainly to control cracking due to plastic shrinkage at an early age. This paper discusses the influence of water-cement ratio, cement-sand ratio and amount of polypropylene fibres in the plastic shrinkage of mortars. A prediction method, based on a factorial design of experiments, is also proposed.