Evidence for the sucrose-binding protein role in carbohydrate metabolism and transport at early developmental stage

Despite the large amount of data regarding sucrose-binding proteins (SBP), their functions remain largely unknown and controversial. In this investigation we performed a detailed temporal and spatial characterization of the phenotypes related to photosynthesis, sucrose exudation and carbohydrate metabolism in SBP antisense plants to gain insights into the physiological role of SBP. Significant reductions in net photosynthesis and in stomatal conductance were observed in the SBP antisense lines but were restricted to the vegetative phase, and persisted during a daily time course at this phase. Photosynthesis was saturated at a substantially lower irradiance in source leaves of the antisense lines, suggesting that light utilization is decreased in these plants. A slight reduction in soluble sugars was observed throughout the development of source leaves, partially overlapping a decrease in sucrose synthase activity (EC 2.4.1.13); whereas a transient increase in starch and adinosine diphosphate (ADP)-glucose pyrophosphorylase activity (EC 2.7.7.27) as well as decreased leaf sucrose exudation were detected in the beginning of the vegetative phase. These changes in source leaves were accompanied by reductions in sucrose and starch in sink leaves, hexoses and sucrose in roots and hexoses in shoot apex, which were observed before the occurrence of a significant reduction in height and in leaf number in the transgenic lines. These alterations in growth parameters did not persist throughout the development, but were associated with a delay in flowering time and leaf senescence in the SBP antisense lines. A likely involvement of SBP in sink strength is discussed.     

IL-17RA Signaling Reduces Inflammation and Mortality during Trypanosoma cruzi Infection by Recruiting Suppressive IL-10-Producing Neutrophils

Members of the IL-17 cytokine family play an important role in protection against pathogens through the induction of different effector mechanisms. We determined that IL-17A, IL-17E and IL-17F are produced during the acute phase of T. cruzi infection. Using IL-17RA knockout (KO) mice, we demonstrate that IL-17RA, the common receptor subunit for many IL-17 family members, is required for host resistance during T. cruzi infection. Furthermore, infected IL-17RA KO mice that lack of response to several IL-17 cytokines showed amplified inflammatory responses with exuberant IFN-γ and TNF production that promoted hepatic damage and mortality. Absence of IL-17RA during T. cruzi infection resulted in reduced CXCL1 and CXCL2 expression in spleen and liver and limited neutrophil recruitment. T. cruzi-stimulated neutrophils secreted IL-10 and showed an IL-10-dependent suppressive phenotype in vitro inhibiting T-cell proliferation and IFN-γ production. Specific depletion of Ly-6G+ neutrophils in vivo during T. cruzi infection raised parasitemia and serum IFN-γ concentration and resulted in increased liver pathology in WT mice and overwhelming wasting disease in IL-17RA KO mice. Adoptively transferred neutrophils were unable to migrate to tissues and to restore resistant phenotype in infected IL-17RA KO mice but migrated to spleen and liver of infected WT mice and downregulated IFN-γ production and increased survival in an IL-10 dependent manner. Our results underscore the role of IL-17RA in the modulation of IFN-γ-mediated inflammatory responses during infections and uncover a previously unrecognized regulatory mechanism that involves the IL-17RA-mediated recruitment of suppressive IL-10-producing neutrophils.     

Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems

Multidrug efflux pumps have emerged as relevant elements in the intrinsic and acquired antibiotic resistance of bacterial pathogens. In contrast with other antibiotic resistance genes that have been obtained by virulent bacteria through horizontal gene transfer, genes coding for multidrug efflux pumps are present in the chromosomes of all living organisms. In addition, these genes are highly conserved (all members of the same species contain the same efflux pumps) and their expression is tightly regulated. Together, these characteristics suggest that the main function of these systems is not resisting the antibiotics used in therapy and that they should have other roles relevant to the behavior of bacteria in their natural ecosystems. Among the potential roles, it has been demonstrated that efflux pumps are important for processes of detoxification of intracellular metabolites, bacterial virulence in both animal and plant hosts, cell homeostasis and intercellular signal trafficking.     

Glutamine homeostasis and mitochondrial dynamics

Glutamine is a multifaceted amino acid that plays key roles in many metabolic pathways and also fulfils essential signaling functions. Although classified as non-essential, recent evidence suggests that glutamine is a conditionally essential amino acid in several physiological situations. Glutamine homeostasis must therefore be exquisitely regulated and mitochondria represent a major site of glutamine metabolism in numerous cell types. Glutaminolysis is mostly a mitochondrial process with repercussions in organelle structure and dynamics suggesting a tight and mutual control between mitochondrial form and cell bioenergetics. In this review we describe an updated account focused on the critical involvement of glutamine in oxidative stress, mitochondrial dysfunction and tumour cell proliferation, with special emphasis in the initial steps of mitochondrial glutamine pathways: transport into the organelle and hydrolytic deamidation through glutaminase enzymes. Some controversial issues about glutamine catabolism within mitochondria are also reviewed.     

Conformational changes in human integrin alphaIIbbeta3 after platelet activation, monitored by FRET

Integrin alpha(IIb)beta(3), an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms. Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However, consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S-S reduction models), which require further experimental tests at the cell level to become established facts. Here, we tested the proposed structural rearrangements undergone by integrin alpha(IIb)beta(3) after cell activation, by using F?rster-type fluorescence resonance energy transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal antibodies directed to the betaA domain of the beta(3) subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the alpha(IIb) subunit (acceptor, Cy3-M3 Fab or Cy3-M10 Fab). The FRET efficiencies observed after ADP or TRAP platelet activation changed less than 20% from the resting values, showing that the distance between the labeled Fab fragments changes only modestly after platelet activation by physiological agonists. This observation is consistent with a conformational model of the activated integrin in the cell less extended than in the switchblade model.     

Angiotensin II Signaling in Human Preadipose Cells: Participation of ERK1,2-Dependent Modulation of Akt

The renin-angiotensin system expressed in adipose tissue has been implicated in the modulation of adipocyte formation, glucose metabolism, triglyceride accumulation, lipolysis, and the onset of the adverse metabolic consequences of obesity. As we investigated angiotensin II signal transduction mechanisms in human preadipose cells, an interplay of extracellular-signal-regulated kinases 1 and 2 (ERK_1,2) and Akt/PKB became evident. Angiotensin II caused attenuation of phosphorylated Akt (p-Akt), at serine 473; the p-Akt/Akt ratio decreased to 0.5±0.2-fold the control value without angiotensin II (p<0.001). Here we report that the reduction of phosphorylated Akt associates with ERK_1,2 activities. In the absence of angiotensin II, inhibition of ERK_1,2 activation with U0126 or PD98059 resulted in a 2.1±0.5 (p<0.001) and 1.4±0.2-fold (p<0.05) increase in the p-Akt/Akt ratio, respectively. In addition, partial knockdown of ERK₁ protein expression by the short hairpin RNA technique also raised phosphorylated Akt in these cells (the p-Akt/Akt ratio was 1.5±0.1-fold the corresponding control; p<0.05). Furthermore, inhibition of ERK_1,2 activation with U0126 prevented the reduction of p-Akt/Akt by angiotensin II. An analogous effect was found on the phosphorylation status of Akt downstream effectors, the forkhead box (Fox) proteins O1 and O4. Altogether, these results indicate that angiotensin II signaling in human preadipose cells involves an ERK_1,2-dependent attenuation of Akt activity, whose impact on the biological functions under its regulation is not fully understood.