The effects of Chronic Lycopene Supplementation on Exercise-Induced Oxidative Stress and Glucose Homeostasis.

Lycopene can exert antioxidant effects against peripheral and cellular oxidative stress and may be associated with reduced diabetic risk. Conversely, exercise-induced free radicals are thought to underpin many of the desirable whole-body adaptations following training and the use of antioxidants within the exercise model remains debatable. PURPOSE: To investigate the effect of lycopene supplementation on oxidative stress and glucose homeostasis following acute aerobic exercise. METHOD: Twenty-eight (n=28) apparently healthy male volunteers were recruited (age 24 �� 4 years; weight 78 �� 10 kg; height 178 �� 8 cm; 2max 40 �� 7 ml��kg-1 ��min-1 ) in a randomised, single blind, placebo-controlled study. Participants were required to attend the Laboratory on two occasions: prior to and following 6 weeks of supplementation of either 10mg lycopene (LG; n=15) or placebo (PG; n=13) followed by a bout of acute exercise for one hour at 65% 2max. Exogenous glucose oxidation was then measured on an isotope ratio mass spectrometer in a sub-group of participants (n=14) following exercise, by administration of a standard oral glucose tolerance test (OGTT; 75g glucose). Venous blood samples were drawn for measurement of oxidative stress parameters, plasma glucose and insulin. RESULTS: Plasma lycopene increased in LG only (0.01 �� 0.004 vs.0.02 �� 0.007 ��mol/L; P <0.05) following supplementation and remained elevated post exercise compared to PG (0.01 �� 0.004 vs. 0.02 �� 0.009 ��mol/L; P <0.05). There were no changes in other markers of oxidative stress (SOD, LOOHs, F2 ISP and Alkoxyl radical) either between or within the trials, (P >0.05, respectively). A main effect for an increase in insulin was observed two hours post OGTT in the sub-groups (Pooled data, P <0.05) but trends in the HOMA scores were evident with a 57% increase for LG (2.20 �� 1.84 vs. 5.14 �� 2.5; P >0.05) and an 11% decrease for PG (2.17 �� 1.06 vs. 1.94 �� 1.53; P >0.05). No change in plasma glucose was detected at any point, or after the OGTT (P >0.05). CONCLUSION: In healthy males, lycopene supplementation had no effect on post exercise levels of ROS or markers of lipid peroxidation, despite an increase in plasma lycopene. However, lycopene supplementation may affect post exercise insulin sensitivity in response to glucose consumption, but further parallel research is required.

CaV3.1 T-Type Ca2+ Channels Contribute to Myogenic Signalling in Rat Retinal Arterioles

Purpose: Although L-type Ca2+ channels are known to play a key role in the myogenic reactivity of retinal arterial vessels, the involvement of other types of voltage-gated Ca2+ channels in this process remains unknown. In the present study we have investigated the contribution of T-type Ca2+ channels to myogenic signalling in arterioles of the rat retinal microcirculation. <br/><br/>Methods: Confocal immunolabelling of wholemount preparations was used to investigate the localisation of CaV3.1-3 channels in retinal arteriolar smooth muscle cells. T-type currents and the contribution of T-type channels to myogenic signalling were assessed by whole-cell patch-clamp recording and pressure myography of isolated retinal arteriole segments. <br/><br/>Results: Strong immunolabelling for CaV3.1 was observed on the plasma membrane of retinal arteriolar smooth muscle cells. In contrast, no expression of CaV3.2 or CaV3.3 could be detected in retinal arterioles, although these channels were present on glial cell end feet surrounding the vessels and retinal ganglion cells, respectively. TTA-A2 sensitive T-type currents were recorded in retinal arteriolar myocytes with biophysical properties distinct from those of the L-type currents present in these cells. Inhibition of T-type channels using TTA-A2 or ML-218 dilated isolated, myogenically active, retinal arterioles. <br/><br/>Conclusions: CaV3.1 T-type Ca2+ channels are functionally expressed on arteriolar smooth muscle cells of retinal arterioles and play an important role in myogenic signalling in these vessels. The work has important implications concerning our understanding of the mechanisms controlling blood flow autoregulation in the retina and its disruption during ocular disease.

Abnormal alterations in the Ca2+/CaV1.2/calmodulin/caMKII signaling pathway in a tremor rat model and in cultured hippocampal neurons exposed to Mg2+-free solution

Voltage-dependent calcium channels (VDCCs) are key elements in epileptogenesis. There are several binding-sites linked to calmodulin (CaM) and several potential CaM-dependent protein kinase II (CaMKII)-mediated phosphorylation sites in CaV1.2. The tremor rat model (TRM) exhibits absence���like seizures from 8 weeks of age. The present study was performed to detect changes in the Ca2+/CaV1.2/CaM/CaMKII pathway in TRMs and in cultured hippocampal neurons exposed to Mg2+���free solution. The expression levels of CaV1.2, CaM and phosphorylated CaMKII (p���CaMKII; Thr���286) in these two models were examined using immunofluorescence and western blotting. Compared with Wistar rats, the expression levels of CaV1.2 and CaM were increased, and the expression of p���CaMKII was decreased in the TRM hippocampus. However, the expression of the targeted proteins was reversed in the TRM temporal cortex. A significant increase in the expression of CaM and decrease in the expression of CaV1.2 were observed in the TRM cerebellum. In the cultured neuron model, p���CaMKII and CaV1.2 were markedly decreased. In addition, neurons exhibiting co���localized expression of CaV1.2 and CaM immunoreactivities were detected. Furthermore, intracellular calcium concentrations were increased in these two models. For the first time, o the best of our knowledge, the data of the present study suggested that abnormal alterations in the Ca2+/CaV1.2/CaM/CaMKII pathway may be involved in epileptogenesis and in the phenotypes of TRMs and cultured hippocampal neurons exposed to Mg2+���free solution.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

<p>Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.</p>

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

<p>Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.</p>

Estudo da interac����o de complexos de van��dio com a ca2+-atpase de ret��culo sarcoplasm��tico: an��lise estrutural e funcional

A bomba de c��lcio de ret��culo sarcoplasm��tico �� uma das prote��nas mais extensivamente estudadas, capaz de interagir com v��rias esp��cies e compostos de van��dio. Combinando-se estudos de fluoresc��ncia com ensaios cin��ticos de transporte e liga����o de 45Ca �� ATPase, avaliou-se o efeito de tr��s complexos de van��dio na fun����o bioenerg��tica e estrutural de Ca2+-ATPase. Demonstrou-se que concentra����es pr��ximas dos valores de IC50 (para a hidr��lise de ATP) de BMOV-V(IV) e de PDC-V(V) n��o inibem significativamente a acumula����o de 45Ca por sarcoves��culas. Por outro lado, o complexo PDC-V(V) mostrou ser capaz de estimular a liga����o de 45Ca ao ret��culo sarcoplasm��tico, sugerindo que este complexos pode interagir com o dom��nio de liga����o de c��lcio �� bomba. V��rios estudos de fluoresc��ncia mostraram que BMOV-V(IV) e PDC-V(V) poder��o ser capazes de induzir a conforma����o E2 (tal como solu����es de ���decavanadato��� e de ���monovanadato���) e E1 de Ca2+-ATPase (tal como solu����es de ���metavanadato���), respectivamente. Apesar de o composto HAIDA-V(IV) previlegiar a conforma����o E1 (devido �� sua elevada afinidade para i��es Ca2+), inibiu significativamente o transporte e a liga����o de 45Ca, o que sugere que possa interagir com os locais de uni��o de c��lcio. Os resultados obtidos s��o consistentes com a forma����o de um aducto entre o composto de van��dio e a prote��na, o que sugere um efeito na homeostasia intracelular de c��lcio, nos sistemas de contrac����o muscular e, inclusive, nas vias de ac����o de insulina. Cada um dos tr��s complexos promoveu respostas distintas na Ca2+-ATPase, sugerindo uma evidencia de actividades biol��gicas diversas em fun����o da esp��cie qu��mica de V e do ambiente de coordena����o.

Iron homeostasis in immune mononuclear cells : a potential role in a atherogenesis

Tese de doutoramento, Biologia (Biologia-Molecular), Universidade de Lisboa, Faculdade de Ci��ncias, 2015

The ubiquitin editing enzyme A20 maintains immune homeostasis and prevents autoimmunity

Dissertation presented to obtain the Doctorate degree (Ph.D.) in Biology at Instituto de Tecnologia Qu��mica e Biol��gica da Universidade Nova de Lisboa

Novel insights into plant vascular development: disclosing a mechanism to maintain thermospermine homeostasis in the xylem

Dissertation presented to obtain the Ph.D degree in Biology

B and T lymphocyte attenuator regulates CD8+ T cell-intrinsic homeostasis and memory cell generation.

B and T lymphocyte attenuator (BTLA) is a negative regulator of T cell activation, but its function in vivo is not well characterized. Here we show that mice deficient in full-length BTLA or its ligand, herpesvirus entry mediator, had increased number of memory CD8(+) T cells. The memory CD8(+) T cell phenotype resulted from a T cell-intrinsic perturbation of the CD8(+) T cell pool. Naive BTLA-deficient CD8(+) T cells were more efficient than wild-type cells at generating memory in a competitive antigen-specific system. This effect was independent of the initial expansion of the responding antigen-specific T cell population. In addition, BTLA negatively regulated antigen-independent homeostatic expansion of CD4(+) and CD8(+) T cells. These results emphasize two central functions of BTLA in limiting T cell activity in vivo.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Molecular mechanisms for the regulation of skin homeostasis

L'ubiquitination est une modification des prot��ines conserv��e, consistant en l'addition de r��sidus �� ubiquitine �� et r��gulant le destin cellulaire des prot��ines. La prot��ine �� TRAF-interacting protein �� TRAIP (ou TRIP) est une ligase E3 qui catalyse l'��tape finale de l'ubiquitination. TRAIP est conserv�� dans l'��volution et est n��cessaire au d��veloppement des organismes puisque l'ablation de TRAIP conduit �� la mort embryonnaire aussi bien de la drosophile que de la souris. De plus, la r��duction de l'expression de TRAIP dans des k��ratinocytes ��pidermiques humains r��prime la prolif��ration cellulaire et induit un arr��t du cycle cellulaire en phase Gl, soulignant le lien ��troit entre TRAIP et la prolif��ration cellulaire. Comme les m��canismes de r��gulation de la prolif��ration jouent un r��le majeur dans l'hom��ostasie de la peau, il est important de caract��riser la fonction de TRAIP dans ces m��canismes. En utilisant des approches in vitro, nous avons d��termin�� que la prot��ine TRAIP est instable, modifi��e par l'addition d'ubiquitine et ayant une demi-vie d'environ 4 heures. Nos analyses ont ��galement r��v��l�� que l'expression de TRAIP est d��pendante du cycle cellulaire, atteignant un pic d'expression en phase G2/M et que l'induction de son expression s'effectue principalement au cours de la transition Gl/S. Nous avons identifi�� le facteur de transcription E2F1 comme en ��tant le responsable, en r��gulant directement le promoteur de TRAIP. Aussi, TRAIP endog��ne ou surexprim��e est surtout localis��e au niveau du nucl��ole, une organelle nucl��aire qui est d��sassembl��e pendant la division cellulaire. Pour examiner la localisation subcellulaire de TRAIP pendant la mitose, nous avons imag�� la prot��ine TRAIP fusionn��e �� une prot��ine fluorescente, �� l'int��rieur de cellules vivantes nomm��es HeLa, �� l'aide d'un microscope confocal. Dans ces conditions, TRAIP est majoritairement localis��e autour des chromosomes en d��but de mitose, puis est arrang��e au niveau de l'ADN chromosomique en fin de mitose. La d��tection de TRAIP endog��ne �� l'aide d'un anticorps sp��cifique a confirm�� cette localisation. Enfin, l'inactivation de TRAIP dans les cellules HeLa par interf��rence ARN a inhib�� leur capacit�� �� s'arr��ter en milieu de mitose. Nos r��sultats sugg��rent que le m��canisme sous-jacent peut ��tre li�� au point de contr��le de l'assemblage du fuseau mitotique. - Ubiquitination of proteins is a post-translational modification which decides the cellular fate of the protein. The TRAF-interacting protein (TRAIP, TRIP) functions as an E3 ubiquitin ligase mediating addition of ubiquitin moieties to proteins. TRAIP interacts with the deubiquitinase CYLD, a tumor suppressor whose functional inactivation leads to skin appendage tumors. TRAIP is required for early embryonic development since removal of TRAIP either in Drosophila or mice by mutations or knock��out is lethal due to aberrant regulation of cell proliferation and apoptosis. Furthermore, shRNA- mediated knock-down of TRAIP in human epidermal keratinocytes (HEK) repressed cell proliferation and induced a Gl/S phase block in the cell cycle. Additionally, TRAIP expression is strongly down- regulated during keratinocyte differentiation supporting the notion of a tight link between TRAIP and cell proliferation. We thus examined the biological functions of TRAIP in epithelial cell proliferation. Using an in vitro approach, we could determine that the TRAIP protein is unstable, modified by addition of ubiquitin moieties after translation and exhibits a half-life of 3.7+/-1-6 hours. Our analysis revealed that the TRAIP expression is modulated in a cell-cycle dependent manner, reaching a maximum expression level in G2/M phases. In addition, the expression of TRAIP was particularly activated during Gl/S phase transition and we could identify the transcription factor E2F1 as an activator of the TRAIP gene promoter. Both endogenous and over-expressed TRAIP mainly localized to the nucleolus, a nuclear organelle which is disassembled during cell division. To examine the subcellular localization of TRAIP during M phase, we performed confocal live-cell imaging of a functional fluorescent protein TRAIP-GFP in HeLa cells. TRAIP was distributed in the cytoplasm and accumulated around mitotic chromosomes in pro- and meta-phasic cells. TRAIP was then confined to chromosomal DNA location in anaphase and later phases of mitosis. Immune-detection of endogenous TRAIP protein confirmed its particular localization in mitosis. Finally, inactivating TRAIP expression in HeLa cells using RNA interference abrogated the cells ability to stop or delay mitosis progression. Our results suggested that TRAIP may involve the spindle assembly checkpoint.

A gene knockout approach in mice to identify glucose sensors controlling glucose homeostasis.

A key aspect of glucose homeostasis is the constant monitoring of blood glucose concentrations by specific glucose sensing units. These sensors, via stimulation of hormone secretion and activation of the autonomic nervous system (ANS), regulate tissue glucose uptake, utilization or production. The best described glucose detection system is that of the pancreatic beta-cells which controls insulin secretion. Secretion of other hormones, in particular glucagon, and activation of the ANS, are regulated by glucose through sensing mechanisms which are much less well characterized. Here I review some of the studies we have performed over the recent years on a mouse model of impaired glucose sensing generated by inactivation of the gene for the glucose transporter GLUT2. This transporter catalyzes glucose uptake by pancreatic beta-cells, the first step in the signaling cascade leading to glucose-stimulated insulin secretion. Inactivation of its gene leads to a loss of glucose sensing and impaired insulin secretion. Transgenic reexpression of the transporter in GLUT2/beta-cells restores their normal secretory function and rescues the mice from early death. As GLUT2 is also expressed in other tissues, these mice were then studied for the presence of other physiological defects due to absence of this transporter. These studies led to the identification of extra-pancreatic, GLUT2-dependent, glucose sensors controlling glucagon secretion and glucose utilization by peripheral tissues, in part through a control of the autonomic nervous system.