TCR- and CD28-mediated recruitment of phosphodiesterase 4 to lipid rafts potentiates TCR signaling

Ligation of the TCR along with the coreceptor CD28 is necessary to elicit T cell activation in vivo, whereas TCR triggering alone does not allow a full T cell response. Upon T cell activation of human peripheral blood T cells, we found that the majority of cAMP was generated in T cell lipid rafts followed by activation of protein kinase A. However, upon TCR and CD28 coligation, beta-arrestin in complex with cAMP-specific phosphodiesterase 4 (PDE4) was recruited to lipid rafts which down-regulated cAMP levels. Whereas inhibition of protein kinase A increased TCR-induced immune responses, inhibition of PDE4 blunted T cell cytokine production. Conversely, overexpression of either PDE4 or beta-arrestin augmented TCR/CD28-stimulated cytokine production. We show here for the first time that the T cell immune response is potentiated by TCR/CD28-mediated recruitment of PDE4 to lipid rafts, which counteracts the local, TCR-induced production of cAMP. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a coreceptor stimulus.     

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.     

Molecular determinants of modulation of CaV2.1 channels by visinin-like protein 2

CaV2.1 channels, which conduct P/Q-type Ca2+ currents, initiate synaptic transmission at most synapses in the central nervous system. Ca2+/calmodulin-dependent facilitation and inactivation of these channels contributes to short-term facilitation and depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin (CaM) from its binding site, differentially regulate CaV2.1 channels, and contribute to the diversity of short-term synaptic plasticity. The neuronal calcium sensor protein visinin-like protein 2 (VILIP-2) inhibits inactivation and enhances facilitation of CaV2.1 channels. Here we examine the molecular determinants for differential regulation of CaV2.1 channels by VILIP-2 and CaM by construction and functional analysis of chimeras in which the functional domains of VILIP-2 are substituted in CaM. Our results show that the N-terminal domain, including its myristoylation site, the central α-helix, and the C-terminal lobe containing EF-hands 3 and 4 of VILIP-2 are sufficient to transfer its regulatory properties to CaM. This regulation by VILIP-2 requires binding to the IQ-like domain of CaV2.1 channels. Our results identify the essential molecular determinants of differential regulation of CaV2.1 channels by VILIP-2 and define the molecular code that these proteins use to control short-term synaptic plasticity.     

Extending body space in immersive virtual reality: a very long arm illusion

Recent studies have shown that a fake body part can be incorporated into human body representation through synchronous multisensory stimulation on the fake and corresponding real body part - the most famous example being the Rubber Hand Illusion. However, the extent to which gross asymmetries in the fake body can be assimilated remains unknown. Participants experienced, through a head-tracked stereo head-mounted display a virtual body coincident with their real body. There were 5 conditions in a between-groups experiment, with 10 participants per condition. In all conditions there was visuo-motor congruence between the real and virtual dominant arm. In an Incongruent condition (I), where the virtual arm length was equal to the real length, there was visuo-tactile incongruence. In four Congruent conditions there was visuo-tactile congruence, but the virtual arm lengths were either equal to (C1), double (C2), triple (C3) or quadruple (C4) the real ones. Questionnaire scores and defensive withdrawal movements in response to a threat showed that the overall level of ownership was high in both C1 and I, and there was no significant difference between these conditions. Additionally, participants experienced ownership over the virtual arm up to three times the length of the real one, and less strongly at four times the length. The illusion did decline, however, with the length of the virtual arm. In the C2-C4 conditions although a measure of proprioceptive drift positively correlated with virtual arm length, there was no correlation between the drift and ownership of the virtual arm, suggesting different underlying mechanisms between ownership and drift. Overall, these findings extend and enrich previous results that multisensory and sensorimotor information can reconstruct our perception of the body shape, size and symmetry even when this is not consistent with normal body proportions.     

Exercise as a model to study redox homeostasis in blood: the effect of protocol and sampling point

Twenty males ran either on a level treadmill (nonmuscle-damaging condition) or on a downhill treadmill (muscle-damaging condition). Blood and urine samples were collected before and after exercise (immediately after, 1h, 4h, 24h, 48h, and 96h). The following assays were performed: F(2)-isoprostanes in urine, protein carbonyls in plasma, glutathione, superoxide dismutase, glutathione peroxidase, and catalase in erythrocytes. The main finding was that monophasic redox responses were detected after nonmuscle-damaging exercise compared to the biphasic responses detected after muscle-damaging exercise. Based on these findings, muscle-damaging exercise may be a more appropriate experimental model to induce physiological oxidative stress.     

Levels of angiopoietins 1 and 2 in induced sputum supernatant in patients with COPD

Pathological features of chronic obstructive pulmonary disease (COPD) include lung vascular remodeling and angiogenesis. Angiopoietin-1 (Ang-1), is an essential mediator of angiogenesis by establishing vascular integrity, whereas angiopoietin-2 (Ang-2) acts as its natural inhibitor. We determined the levels of angiopoietins in sputum supernatants of patients with COPD and investigated their possible association with mediators and cells involved in the inflammatory and remodeling process. Fifty-nine patients with COPD, 25 healthy smokers and 20 healthy non-smokers were studied. All subjects underwent lung function tests, sputum induction for cell count identification and Ang-1, Ang-2, VEGF, TGF-β1, MMP-2, LTB4, IL-8, albumin measurement in sputum supernatants. Airway vascular permeability (AVP) index was also assessed. Ang-2 levels were significantly higher in patients with COPD compared to healthy smokers and healthy non-smokers [median, interquartile ranges pg/ml, 267 (147-367) vs. 112 (67-171) and 98 (95-107), respectively; p<0.001]. Regression analysis showed a significant association between Ang-2 levels and AVP index, VEGF, IL-8 and MMP-2 levels in COPD, the strongest being with VEGF. Our results indicate that induced sputum Ang-2 levels are higher in COPD compared to healthy smokers and healthy non-smokers. Moreover, Ang-2 is associated with AVP, IL-8, MMP-2, and VEGF, indicating a possible role for Ang-2 in the pathogenesis of the disease.     

Diversity of spiders and orthopterans respond to intra-seasonal and spatial environmental changes

Our understanding of arthropod responses to environmental pressures is limited, especially for the poorly studied Mediterranean region. In the light of likely further environmental change and the need for protocols for rapid biodiversity assessment, we measured how the abundance and species richness of two taxa, ground spiders and Orthoptera, belonging to different functional groups, fluctuates intra- seasonally (early-mid-late summer) and across habitat types (grasslands, maquis, forests). We also tested their surrogate value. Spiders were found to have higher species richness and abundance almost throughout the investigation. Orthoptera had lower species richness and abundance in forests compared to grasslands and maquis, while no significant difference between habitats was revealed for spiders. Early-summer was the richest period for spiders while mid-summer was the richest for Orthoptera. Canopy cover was found to significantly influence community composition of both groups, while herb height and cover of stones was a determinant factor for Orthoptera only. There was a significant congruence between the two groups and Orthoptera provided the best complementary network. Our results show that diversity patterns of both spiders and Orthoptera are sensitive to environmental changes even over short time-scales (e.g. within the summer period) and space (e.g. across different habitat types), suggesting that small inexpensive experimental designs may still reveal community dynamics. For conservation purposes, we advise a focus on variables regulating habitat heterogeneity and microhabitat characteristics. We provide a list of the most influential species and propose the most effective network for obtaining information on the local fauna.     

Identification of Novel Thymic Epithelial Cell Subsets Whose Differentiation Is Regulated by RANKL and Traf6

Thymic epithelial cells (TECs) are critical for the normal development and function of the thymus. Here, we examined the developmental stages of TECs using quantitative assessment of the cortical and medullary markers Keratin 5 and Keratin 8 (K5 and K8) respectively, in normal and gain/loss of function mutant animals. Gain of function mice overexpressed RANKL in T cells, whereas loss of function animals lacked expression of Traf6 in TECs (Traf6ΔTEC). Assessment of K5 and K8 expression in conjunction with other TEC markers in wild type mice identified novel cortical and medullary TEC populations, expressing different combinations of these markers. RANKL overexpression led to expansion of all medullary TECs (mTECs) and enlargement of the thymic medulla. This in turn associated with a block in thymocyte development and loss of CD4⁺CD8⁺, CD4⁺ and CD8⁺ thymocytes. In contrast, Traf6 deletion inhibited the production of most TEC populations including cortical TECs (cTECs), defined by absence of UEA-1 binding and LY51 expression, but had no apparent effect on thymocyte development. These results reveal a large degree of heterogeneity within the TEC compartment and the existence of several populations exhibiting concomitant expression of cortical, medullary and epithelial markers and whose production is regulated by RANKL and Traf6.     

The role of heparins and nano-heparins as therapeutic tool in breast cancer

Glycosaminoglycans are integral part of the dynamic extracellular matrix (ECM) network that control crucial biochemical and biomechanical signals required for tissue morphogenesis, differentiation, homeostasis and cancer development. Breast cancer cells communicate with stromal ones to modulate ECM mainly through release of soluble effectors during cancer progression. The intracellular cross-talk between cell surface receptors and estrogen receptors is important for the regulation of breast cancer cell properties and production of ECM molecules. In turn, reorganized ECM-cell surface interface modulates signaling cascades, which regulate almost all aspects of breast cell behavior. Heparan sulfate chains present on cell surface and matrix proteoglycans are involved in regulation of breast cancer functions since they are capable of binding numerous matrix molecules, growth factors and inflammatory mediators thus modulating their signaling. In addition to its anticoagulant activity, there is accumulating evidence highlighting various anticancer activities of heparin and nano-heparin derivatives in numerous types of cancer. Importantly, heparin derivatives significantly reduce breast cancer cell proliferation and metastasis in vitro and in vivo models as well as regulates the expression profile of major ECM macromolecules, providing strong evidence for therapeutic targeting. Nano-formulations of the glycosaminoglycan heparin are possibly novel tools for targeting tumor microenvironment. In this review, the role of heparan sulfate/heparin and its nano-formulations in breast cancer biology are presented and discussed in terms of future pharmacological targeting.     

Peptide Finder: mapping measured molecular masses to peptides and proteins

The identification of unknown amino acid sequences of peptides as well as protein identification is of great significance in proteomics. Here, we present a publicly available web application that facilitates a high resolution mapping of measured molecular masses to peptides and proteins, irrespectively of the enzyme/digestion method used. Furthermore, multi-filtering may be applied in terms of measured mass tolerance, molecular mass and isoelectric point range as well as pattern matching to refine the results. This approach serves complementary to the existing solutions for protein identification and gives insights in novel peptides discovery and protein identification at the cases where the identification scores from the other approaches may be below significance threshold. Peptide Finder has been proven useful in proteomics procedures with experimental data from MALDI-TOF. AVAILABILITY: Peptide Finder web-application is available at http://bioserver-1.bioacademy.gr/Bioserver/PeptideFinder/.