944 resultados para Intracellular Ca2 store
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El Estrs de Retculo Endoplsmico (RE) es inducido por la acumulacin de protenas sin plegar en el lumen de la organela. Esto se puede observar en diversas situaciones fisio-patolgicas como durante una infeccin viral o en proceso isqumico. Adems, contribuye a la base molecular de numerosas enfermedades ya sea ndole metablico (Fibrosis qustica o Diabetes Miellitus) o neurodegenerativas como mal de Alzheimer o Parkinson (Mutat Res, 2005, 569). Para restablecer la homeostasis en la organela se activa una seal de transduccin (UPR), cuya respuesta inmediata es la atenuacin de la sntesis de protena debido a la fosforilacin de subunidad alpha del factor eucaritico de iniciacin de translacin (eIF2) va PERK. Esta es una protena de membrana de RE que detecta estrs. Bajo condiciones normales, PERK est inactiva debido a la asociacin de su dominio luminar con la chaperona BIP (Nat Cell Biol, 2000, 2: 326). Frente a una situacin de estrs, la chaperona se disocia causando desinhibicin. Recientemente, (Plos One 5: e11925) se observ, bajo condiciones de estrs, un aumento de Ca2+ citoslico y un rpido incremento de la expresin de calcineurina (CN), una fosfatasa citoslica dependiente de calcio, heterodimrica formada por una subunidad cataltica (CN-A) y una regulatoria (CN-B). Adems, CN interacciona, sin intermediarios, con el dominio citoslico de PERK favoreciendo su trans-autofosforilacin. Resultados preliminares indican que, astrocitos CNA-/- exhibieron, en condiciones basales, un mayor nmero de clulas muertas y de niveles de eIF2 fosforilado que los astrocitos CNA-/-. Hiptesis: CNA/B interacciona con PERK cuando el Ca2+ citoslico esta incrementado luego de haberse inducido Estrs de RE, lo cual promueve dimerizacin y auto-fosforilacin de la quinasa, acentundose as la fosforilacin de eIF2 e inhibicin de la sntesis de protenas. Esta activacin citoslica de PERK colaborara con la ya descrita, desinhibicin luminal llevada cabo por BIP. Cuando el Ca2+ citoslico retorna a los niveles basales, PERK fosforila a CN, reduciendo su afinidad de unin y disocindose el complejo CN/PERK. Objetivo general: Definir las condiciones por las cuales CN interacciona con PERK y regula la fosforilacin de eIF2 e inhibicin de la sntesis de protena. Objetivos especficos: I-Estudiar la diferencia de afinidades y dependencia de Ca2+, de las dos isoformas de CN ( y ) en su asociacin con PERK. Adems verificar la posible participacin de la subunidad B de CN en esta interaccin. II-Determinar si la auto-fosforilacin de PERK es diferencialmente regulada por las dos isoformas de CN. III-Discernir la relacin del estado de fosforilacin de CN con su unin a PERK. IV-Determinar efectos fisiolgicos de la interaccin de CN-PERK durante la respuesta de Estrs de RE. Para llevar a cabo este proyecto se realizarn experimentos de biologa molecular, interaccin protena-protena, ensayos de fosforilacin in vitro y un perfil de polisoma con astrocitos CNA-/- , CNA-/- y astrocitos controles. Se espera encontrar una mayor afinidad de unin a PERK de la isoforma de CN y en condiciones donde la concentracin de Ca2+ sea del orden micromolar e imite niveles del in durante un estrs. Con respecto al estado de fosforilacin de CN, debido a los resultados preliminares, donde solo se la encontr fosforilada en condiciones basales, se piensa que CN podra interactuar con mayor afinidad con PERK cuando CN se encuentre desfosforilada. Por ltimo, se espera encontrar un aumento de eIF2 fosforilado y una acentuacin de la atenuacin de la sntesis de protena como consecuencia de la mayor activacin de PERK por su asociacin con la isoforma de CN en astrocitos donde el Estrs de RE se indujo por privacin de oxigeno y glucosa. Estos experimentos permitirn avanzar en el estudio de una nueva funcin citoprotectora de CN recientemente descrita por nuestro grupo de trabajo y sus implicancias en un modelo de isquemia. The accumulation of unfolded proteins into the Endoplasmic Reticulum (ER) activates a signal transduction cascade called Unfolding Protein Response (UPR), which attempts to restore homeostasis in the organelle. (PKR)-like-ER kinase (PERK) is an early stress response transmembrane protein that is generally inactive due to its association with the chaperone BIP. During ER stress, BIP is tritrated by the unfolded protein, leading PERK activation and phosphorylation of eukaryotic initiation factor-2 alpha (eIF2alpha), which attenuates protein sntesis. If ER damage is too great and homeostasis is not restored within a certain period of time, an apoptotic response is elicited. We recently demonstrated a cytosolic Ca2+ increase in Xenopus oocytes after induce ER stress. Moreover, calcineurin A/B, a an heterotrimeric Ca2+ dependent phosphatases (CN-A/B), associates with PERK increasing its auto-phosphorylation and significantly enhancing cell viability. Preliminary results suggest that, CN-A-/- knockout astrocytes exhibit a significant higher eIF2 phosphorylated level compared to CN-A-/- astrocytes. Our working hypothesis establishes that: CN binds to PERK when cytosolic Ca2+ is initially increased by ER stress, promoting dimerization and autophosphorylation, which leads to phosphorylation of elF2 and subsequently attenuation of protein translation. When cytosolic Ca2+ returns to resting levels, PERK phosphorylates CN, reducing its binding affinity so that the CN/PERK complex dissociates. The goal of this project is to determine the conditions by which CN binding to PERK attenuates protein translation during the ER stress response and subsequently, to determine how the interaction of CN with PERK is terminated when stress is removed. To perform this project is planed to do molecular biology experiments, pull down assays, in vitro phosphorylations and assess overall mRNA translation efficiency doing a polisome profile.
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FUNDAMENTO: Treinamento fsico (TF) aumenta a sensibilidade dos hormnios tireoidianos (HT) e a expresso gnica de estruturas moleculares envolvidas no movimento intracelular de clcio do miocrdio, enquanto a restrio alimentar (RIA) promove efeitos contrrios ao TF. OBJETIVO: Avaliar os efeitos da associao TF e RIA sobre os nveis plasmticos dos HT e a produo de mRNA dos receptores HT e estruturas moleculares do movimento de clcio do miocrdio de ratos. MTODOS: Utilizaram-se ratos Wistar Kyoto divididos em: controle (C, n = 7), RIA (R50, n = 7), exerccio fsico (EX, n = 7) e exerccio fsico + RIA (EX50, n = 7). A RIA foi de 50% e o TF foi natao (1 hora/dia, cinco sesses/semana, 12 semanas consecutivas). Avaliaram-se as concentraes sricas de triiodotironina (T3), tiroxina (T4) e hormnio tireotrfico (TSH). O mRNA da bomba de clcio do retculo sarcoplasmtico (SERCA2a), fosfolamban (PLB), trocador Na+/Ca+2 (NCX), canal lento de clcio (canal-L), rianodina (RYR), calsequestrina (CQS) e receptor de HT (TRα1 e TRβ1) do miocrdio foram avaliados por reao em cadeia da polimerase (PCR) em tempo real. RESULTADOS: RIA reduziu o T4, TSH e mRNA do TRα1 e aumentou a expresso da PLB, NCX e canal-L. TF aumentou a expresso do TRβ1, canal-L e NCX. A associao TF e RIA reduziu T4 e TSH e aumentou o mRNA do TRβ1, SERCA2a, NCX, PLB e correlao do TRβ1 com a CQS e NCX. CONCLUSO: Associao TF e RIA aumentou o mRNA das estruturas moleculares clcio transiente, porm o eixo HT-receptor no parece participar da transcrio gnica dessas estruturas.
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Background: Stress is associated with cardiovascular diseases. Objective: This study aimed at assessing whether chronic stress induces vascular alterations, and whether these modulations are nitric oxide (NO) and Ca2+ dependent. Methods: Wistar rats, 30 days of age, were separated into 2 groups: control (C) and Stress (St). Chronic stress consisted of immobilization for 1 hour/day, 5 days/week, 15 weeks. Systolic blood pressure was assessed. Vascular studies on aortic rings were performed. Concentration-effect curves were built for noradrenaline, in the presence of L-NAME or prazosin, acetylcholine, sodium nitroprusside and KCl. In addition, Ca2+ flux was also evaluated. Results: Chronic stress induced hypertension, decreased the vascular response to KCl and to noradrenaline, and increased the vascular response to acetylcholine. L-NAME blunted the difference observed in noradrenaline curves. Furthermore, contractile response to Ca2+ was decreased in the aorta of stressed rats. Conclusion: Our data suggest that the vascular response to chronic stress is an adaptation to its deleterious effects, such as hypertension. In addition, this adaptation is NO- and Ca2+-dependent. These data help to clarify the contribution of stress to cardiovascular abnormalities. However, further studies are necessary to better elucidate the mechanisms involved in the cardiovascular dysfunction associated with stressors. (Arq Bras Cardiol. 2014; [online].ahead print, PP.0-0)
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Magdeburg, Univ., Fak. fr Naturwiss., Diss., 2009
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Magdeburg, Univ., Fak. fr Verfahrens- und Systemtechnik, Diss., 2015
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Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P(1) residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.
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While genetic polymorphisms play a paramount role in tuberculosis (TB), less is known about their contribution to the severity of diseases caused by other intracellular bacteria and fastidious microorganisms. We searched electronic databases for observational studies reporting on host factors and genetic predisposition to infections caused by intracellular fastidious bacteria published up to 30 May 2014. The contribution of genetic polymorphisms was documented for TB. This includes genetic defects in the mononuclear phagocyte/T helper cell type 1 (Th1) pathway contributing to disseminated TB disease in children and genome-wide linkage analysis (GWAS) in reactivated pulmonary TB in adults. Similarly, experimental studies supported the role of host genetic factors in the clinical presentation of illnesses resulting from other fastidious intracellular bacteria. These include IL-6 -174G/C or low mannose-binding (MBL) polymorphisms, which are incriminated in chronic pulmonary conditions triggered by C.pneumoniae, type 2-like cytokine secretion polymorphisms, which are correlated with various clinical patterns of M.pneumoniae infections, and genetic variation in the NOD2 gene, which is an indicator of tubal pathology resulting from Chamydia trachomatis infections. Monocyte/macrophage migration and T lymphocyte recruitment defects are corroborated to ineffective granuloma formation observed among patients with chronic Q fever. Similar genetic polymorphisms have also been suggested for infections caused by T.whipplei although not confirmed yet. In conclusion, this review supports the paramount role of genetic factors in clinical presentations and severity of infections caused by intracellular fastidious bacteria. Genetic predisposition should be further explored through such as exome sequencing.
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Summary The best described physiological function of low-density lipoproteins (LDL) is to transport cholesterol to target tissues. LDL deliver their cholesterol cargo to cells following their interaction with the LDL receptor. LDL, when their vascular concentrations increase, have also been implicated in pathologies such as atherosclerosis. Among the cell types that are found in blood vessels, endothelial and smooth muscle cells have dominated cellular research on atherosclerotic mechanisms and LDL activation of signaling pathways, while very little is known about adventitial fibroblast activation caused by elevated lipoprotein levels. Since fibroblasts participate in wound repair and since it has recently been recognized that fibroblasts may play pivotal roles in vascular remodeling and repair of injury, we assessed whether lipoproteins affect fibroblast function. We have found that LDL specifically mediate the activation of a class of mitogen-activated protein kinases (MAPKs): the p38 MAPKs. The activation of this pathway in turn modulates cell shape by promoting lamellipodia formation and extensive cell spreading. This is of particular interest because it provides a mechanism by which LDL can promote wound healing or vessel wall remodeling as observed during the development of atherosclerosis. In order to understand the molecular mechanisms by which LDL induce p38 activation we searched for the component in the LDL particle responsible for the induction of this pathway. We found that cholesterol is the major component of lipoprotein particles that mediates their ability to stimulate the p38 MAPK pathway. Furthermore, we investigated the cellular mechanisms underlying the ability of LDL to induce cell shape changes and whether this could participate in wound repair. Our recent data demonstrates that the capacity of LDL to induce fibroblast spreading relies on their ability to stimulate IL-8 secretion, which in turn leads to accelerated wound healing. LDL-induced IL-8 production and subsequent wound closure are impaired upon inhibition of the p38 MAPK pathway indicating that the LDL-induced spreading and accelerated wound sealing rely on the ability of LDL to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Therefore, regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL-cholesterol levels, IL-8 production and extensive remodeling of the vessel wall. Rsum: La fonction physiologique des lipoprotines faible densit (LDL) la mieux dcrite est celle du transport du cholestrol aux tissus cibles. Les LDL livrent leur cargaison de cholestrol aux cellules aprs leur interaction avec le rcepteur au LDL. Une concentration vasculaire des LDL augment est galement implique dans le dveloppement de l'athrosclrose. Parmi les types de cellule prsents dans les vaisseaux sanguins, les cellules endothliales et les cellules du muscle lisse ont domin la recherche cellulaire sur les mcanismes athrosclrotiques et sur l'activation par les LDL des voies de signalisation intracellulaire. A l'inverse peu de choses sont connues sur l'activation des fibroblastes de l'adventice par les lipoprotines. Puisqu'il a t rcemment reconnu que les fibroblastes peuvent jouer un rle central dans la remodlisation vasculaire et la rparation tissulaire, nous avons tudi si les lipoprotines affectent la fonction des fibroblastes. Nous avons constat que les LDL activent spcifiquement une classe de protines kinases: les p38 MAPK (mitogen-activated protein kinases). L'activation de cette voie module son tour la forme de la cellule en favorisant la formation de lamellipodes et l'agrandissement des cellules. Cela a un intrt particulier car il fournit un mcanisme par lequel les LDL peuvent promouvoir la cicatrisation ou la remodlisation des parois vasculaires comme observs lors du dveloppement de l'athrosclrose. Pour comprendre les mcanismes molculaires par lesquels les LDL provoquent l'activation des p38 MAPK, nous avons cherch identifier les composants dans la particule de LDL responsables de l'induction de cette voie. Nous avons constat que le cholestrol est l'lment principal des particules de lipoprotine qui contrle leur capacit stimuler la voie des p38 MAPK. En outre, nous avons examin les mcanismes cellulaires responsables de la capacit des LDL induire des changements dans la forme des cellules. Nos donnes rcentes dmontrent que la capacit des LDL induire l'agrandissement des cellules, ainsi que leur aptitude favoriser la cicatrisation, reposant sur leur capacit stimuler la scrtiond'IL-8. La production d'IL-8 induite par les LDL est bloque par l'inhibition de la voie p38 MAPK, ce qui indique que l'talement des cellules induit par les LDL ainsi que l'acclration de la cicatrisation sont lis la capacit des LDL stimuler la scrtion d'IL8 via l'activation des p38 MAPK. La rgulation de la forme et de la migration des fibroblastes par les lipoprotines peuvent donc participer au dveloppement de l'athrosclrose qui est caractrise par l'augmentation des niveaux de production de LDL-cholestrol et d'IL-8 ainsi que par une remodlisation augmente de la paroi du vaisseau.
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RESUME GRAND PUBLICLe cerveau est compos de diffrents types cellulaires, dont les neurones et les astrocytes. Faute de moyens pour les observer, les astrocytes sont trs longtemps rests dans l'ombre alors que les neurones, bnficiant des outils ad hoc pour tre stimuls et tudis, ont fait l'objet de toutes les attentions. Le dveloppement de l'imagerie cellulaire et des outils fluorescents ont permis d'observer ces cellules non lectriquement excitables et d'obtenir des informations qui laissent penser que ces cellules sont loin d'tre passives et participent activement au fonctionnement crbral. Cette participation au fonctionnement crbral se fait en partie par le biais de la libration de substances neuro-actives (appelles gliotransmetteurs) que les astrocytes librent proximit des synapses permettant ainsi de moduler le fonctionnement neuronal. Cette libration de gliotransmetteurs est principalement cause par l'activit neuronale que les astrocytes sont capables de sentir. Nanmoins, nous savons encore peu de chose sur les proprits prcises de la libration des gliotransmetteurs. Comprendre les proprits spatio-temporelles de cette libration est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information crbrale. En utilisant des outils fluorescents rcemment dvelopps et en combinant diffrentes techniques d'imagerie cellulaire, nous avons pu obtenir des informations trs prcises sur la libration de ces gliotransmetteurs par les astrocytes. Nous avons ainsi confirm que cette libration tait un processus trs rapide et qu'elle tait contrle par des augmentations de calcium locales et rapides. Nous avons galement dcrit une organisation complexe de la machinerie supportant la libration des gliotransmetteurs. Cette organisation complexe semble tre la base de la libration extrmement rapide des gliotransmetteurs. Cette rapidit de libration et cette complexit structurelle semblent indiquer que les astrocytes sont des cellules particulirement adaptes une communication rapide et qu'elles peuvent, au mme titre que les neurones dont elles seraient les partenaires lgitimes, participer la transmission et l'intgration de l'information crbrale.RESUMEDe petites vsicules, les SLMVs ou Synaptic Like MicroVesicles , exprimant des transporteurs vsiculaires du glutamate (VGluTs) et librant du glutamate par exocytose rgule, ont rcemment t dcrites dans les astrocytes en culture et in situ. Nanmoins, nous savons peu de chose sur les proprits prcises de la scrtion de ces SLMVs. Contrairement aux neurones, le couplage stimulusscrtion des astrocytes n'est pas bas sur l'ouverture des canaux calciques membranaires mais ncessite l'intervention de seconds messagers et la libration du calcium par le reticulum endoplasmique (RE). Comprendre les proprits spatio-temporelles de la scrtion astrocytaire est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information crbrale. Nous avons utilis des outils fluorescents rcemment dvelopps pour tudier le recyclage des vsicules synaptiques glutamatergiques comme les colorants styryles et la pHluorin afin de pouvoir suivre la scrtion des SLMVs l'chelle de la cellule mais galement l'chelle des vnements. L'utilisation combine de l'pifluorescence et de la fluorescence onde vanescente nous a permis d'obtenir une rsolution temporelle et spatiale sans prcdent. Ainsi avons-nous confirm que la scrtion rgule des astrocytes tait un processus trs rapide (de l'ordre de quelques centaines de millisecondes). Nous avons dcouvert que cette scrtion est contrle par des augmentations de calcium locales et rapides. Nous avons galement dcrit des compartiments cytosoliques dlimits par le RE proximit de la membrane plasmique et contenant les SLMVs. Cette organisation semble tre la base du couplage rapide entre l'activation des GPCRs et la scrtion. L'existence de compartiments subcellulaires indpendants permettant de contenir les messagers intracellulaires et de limiter leur diffusion semble compenser de manire efficace la nonexcitabilit lectrique des astrocytes. Par ailleurs, l'existence des diffrents pools de vsicules recruts squentiellement et fusionnant selon des modalits distinctes ainsi que l'existence de mcanismes permettant le renouvellement de ces pools lors de la stimulation suggrent que les astrocytes peuvent faire face une stimulation soutenue de leur scrtion. Ces donnes suggrent que la libration de gliotransmetteurs par exocytose rgule n'est pas seulement une proprit des astrocytes en culture mais bien le rsultat d'une forte spcialisation de ces cellules pour la scrtion. La rapidit de cette scrtion donne aux astrocytes toutes les comptences pour pouvoir intervenir de manire active dans la transmission et l'intgration de l'information.ABSTRACTRecently, astrocytic synaptic like microvesicles (SLMVs), that express vesicular glutamate transporters (VGluTs) and are able to release glutamate by Ca2+-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Nevertheless, little is known about the specific properties of regulated secretion in astrocytes. Important differences may exist between astrocytic and neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca2+ from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We took advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses like styryl dyes and pHluorin in order to follow exocytosis and endocytosis of SLMVs at the level of the entire cell or at the level of single event. We combined epifluorescence and total internal reflection fluorescence imaging to investigate, with unprecedented temporal and spatial resolution, the events underlying the stimulus-secretion in astrocytes. We confirmed that exo-endocytosis process in astrocytes proceeds with a time course on the millisecond time scale. We discovered that SLMVs exocytosis is controlled by local and fast Ca2+ elevations; indeed submicrometer cytosolic compartments delimited by endoplasmic reticulum (ER) tubuli reaching beneath the plasma membrane and containing SLMVs. Such complex organization seems to support the fast stimulus-secretion coupling reported here. Independent subcellular compartments formed by ER, SLMVs and plasma membrane containing intracellular messengers and limiting their diffusion seem to compensate efficiently the non-electrical excitability of astrocytes. Moreover, the existence of two pools of SLMVs which are sequentially recruited suggests a compensatory mechanisms allowing the refill of SLMVs and supporting exocytosis process over a wide range of multiple stimuli. These data suggest that regulated secretion is not only a feature of cultured astrocytes but results from a strong specialization of these cells. The rapidity of secretion demonstrates that astrocytes are able to actively participate in brain information transmission and processing.
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The fundamental processes of membrane fission and fusion determine size and copy numbers of intracellular organelles. Although SNARE proteins and tethering complexes mediate intracellular membrane fusion, fission requires the presence of dynamin or dynamin-related proteins. Here we study these reactions in native yeast vacuoles and find that the yeast dynamin homologue Vps1 is not only an essential part of the fission machinery, but also controls membrane fusion by generating an active Qa SNARE-tethering complex pool, which is essential for trans-SNARE formation. Our findings provide new insight into the role of dynamins in membrane fusion by directly acting on SNARE proteins.
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During adult thymus development immature CD4(-)CD8(-) [double-negative (DN)] precursor cells pass through four phenotypically distinct stages defined by expression of CD44 and CD25: CD44(hi)CD25(-) (DN1), CD44(hi)CD25(+) (DN2), CD44(lo)CD25(+) (DN3) and CD44(lo)CD25(-) (DN4). Although it is well established that the TCR beta, gamma and delta genes are rearranged and expressed in association with the CD3 components in DN thymocytes, the precise timing of expression of the TCR and CD3 proteins has not been determined. In this report we have utilized a sensitive intracellular (ic) staining technique to analyze the expression of ic CD3epsilon, TCR beta and TCR gammadelta proteins in immature DN subsets. As expected from previous studies of TCR beta rearrangement and mRNA expression, icTCR beta(+) cells were first detected in the DN3 subset and their proportion increased thereafter. Surprisingly, however, both icCD3epsilon(+) and icTCR gammadelta(+) cells were detected at later stages of development than was predicted by molecular studies. In particular icCD3epsilon protein expression coincided with the transition from the DN2 to DN3 stage of development, whereas icTCR gammadelta protein expression was only detected in a minor subset of DN4 cells. The implications of these findings for alphabeta lineage divergence will be discussed.
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Immunity to intracellular bacteria including Mycobacterium tuberculosis. Mycobacterium leprae, and Listeria monocytogenes depends on specific T cells. Evidence to be described suggests that CD4 (alpha/beta)T cells which interact with each other and with macrophages contribute to acquired resistence against as well as pathogenesis of intracellular bacterial infections.
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Conformational changes of channel activation: Five enhanced green fluorescent protein (EGFP) molecules (green cylinders) were integrated into the intracellular part of the homopentameric ionotropic 5-HT3 receptor. This allowed the detection of extracellular binding of fluorescent ligands (?) to EGFP by FRET, and also enabled the quantification of agonist-induced conformational changes in the intracellular region of the receptor by homo-FRET between EGFPs. The approach opens novel ways for probing receptor activation and functional screening of therapeutic compounds.
