Rôle des récepteurs Toll-like et de la protéine adaptatrice MyD88 dans la régulation de l’hepcidine et le développement des hyposidérémies associées à l’inflammation

Le fer est un oligo-élément nécessaire pour le fonctionnement normal de toutes les cellules de l'organisme et joue un rôle essentiel dans de nombreuses fonctions biologiques. Cependant, le niveau de fer dans le corps doit être bien réglé, sinon la carence en fer entraine des divers états pathologiques tels que l'anémie et la diminution de l’immunité. D'autre part, une surcharge en fer potentialise la multiplication des germes, aggrave l’infection et la formation de radicaux libres ayant des effets toxiques sur les cellules et leurs composants, ce qui favorise les maladies cardio-vasculaires, l'inflammation et le cancer. L'hepcidine (HAMP), un régulateur négatif de l'absorption du fer, induit la dégradation de la ferroportine (FPN), le seul exportateur connu de fer ce qui réduit sa libération par les macrophages et inhibe son absorption gastro-intestinale. HAMP est synthétisé principalement par les hépatocytes, mais aussi par les macrophages. Cependant, il y a très peu de données sur la façon dont HAMP est régulé au niveau des macrophages. Plus récemment, nous avons constaté que l’induction de l’hepcidin dans le foie par le polysaccharide (LPS) est dépendante de la voie de signalisation médiée par « Toll-like receptor 4 » (TLR4). Grâce au TLR4, le LPS induit l'activation des macrophages qui sécrètent de nombreuses différentes cytokines inflammatoires, y compris Interleukine 6 (IL-6), responsable de l'expression de HAMP hépatique. Dans le premier chapitre de la présente étude, nous avons étudié la régulation de HAMP dans la lignée cellulaire macrophagique RAW264.7 et dans les macrophages péritonéaux murins stimulés par différents ligands des TLRs. Nous avons constaté que TLR2 et TLR4 par l'intermédiaire de la protéine adaptatrice « myeloid differentiation primary response gene 88 » (MyD88) activent l'expression de HAMP dans les cellules RAW264.7 et les macrophages péritonéaux sauvages murins, tandis que cette expression a été supprimée dans les macrophages isolés des souris TLR2-/-, TLR4-déficiente ou MyD88-/-. En outre, nous avons constaté que la production d'IL-6 par les cellules RAW264.7 stimulées avec du LPS a été renforcée par l’ajout des quantités élevées de fer dans le milieu de culture. Au cours de l’inflammation, le niveau de HAMP est fortement augmenté. Ainsi, lorsque l'inflammation persiste, l’expression de HAMP continue à être activée par des cytokines pro-inflammatoires conduisant à une hyposidérémie. Malgré que cette dernière soit considérée comme une défense de l'hôte pour priver les micro-organismes de fer, celle ci cause un développement d'anémies nommées anémies des maladies chroniques. Ainsi, dans le deuxième chapitre de la présente étude, nous avons étudié l'implication des TLRs et leurs protéines adaptatrices MyD88 et TIR-domain-containing adapter-inducing interferon-β (TRIF) dans le développement des hyposidérémies. En utilisant des souris déficientes en MyD88 et TRIF, nous avons montré que les voies de signalisations MyD88 et TRIF sont essentielles pour l’induction de HAMP par le LPS. Malgré l'absence de HAMP, les souris déficientes ont été capables de développer une hyposidérémie, mais la réponse des souris déficientes en MyD88 a été très légère, ce qui indique l'exigence de cette protéine pour assurer une réponse maximale au LPS. En outre, nous avons constaté que la signalisation MyD88 est nécessaire pour le stockage du fer au niveau de la rate, ainsi que l'induction de lipocaline 2 (LCN2), qui est une protéine impliquée dans la fixation du fer pour limiter la croissance bactérienne. Indépendamment de MyD88 ou TRIF, l'activation de TLR4 et TLR3 a conduit, au niveau de la rate, à une diminution rapide de l’expression de FPN et du « Human hemochromatosis protein » (HFE) qui est une protéine qui limite la séquestration du fer cellulaire à partir de la circulation. Cependant, malgré cette baisse d’expression, le manque de la signalisation MyD88 a altéré de manière significative la réponse hyposidérémique. En établissant le rôle des TLRs et de la protéine adaptatrice MyD88 dans la diminution du taux du fer sérique au cours de la réponse inflammatoire, nous avons remarqué qu’en réponse au surcharge en fer les souris déficientes en MyD88 accumulent de manière significative plus de fer hépatique par rapport aux souris sauvages, et cela indépendamment des TLRs. Ainsi, dans le troisième chapitre de la présente étude, nous avons étudié le phénotype observé chez les souris déficientes en MyD88. Nous avons trouvé que l'expression de HAMP chez ces souris a été plus faible que celle des souris de type sauvage. Pour cela, nous avons exploré la signalisation à travers la voie du « Bone Morphogenetic Proteins 6 » (BMP6) qui est considérée comme étant la voie fondamentale de la régulation de HAMP en réponse aux concentrations du fer intracellulaires et extracellulaires et nous avons trouvé que l'expression protéique de Smad4, un régulateur positif de l'expression de HAMP, est significativement plus faible chez les souris MyD88-/- par rapport aux souris sauvages. En outre, on a montré que MyD88 interagit avec « mothers against decapentaplegic, Drosophila, homolog 4 » (Smad4) et que cette interaction est essentielle pour l’induction de HAMP à travers la voie BMP6. En conclusion, notre étude montre que l'expression de HAMP dans les macrophages est régulée principalement par TLR2 et TLR4 à travers la voie MyD88 et que l'accumulation du fer dans les macrophages peut affecter les niveaux des cytokines pro-inflammatoires. En outre, nos analyses démontrent que le développement d’hyposidérémie en réponse au LPS se produit par l'intermédiaire d’un mécanisme dépendant de MyD88 qui est dissociée de la production de cytokines et de HAMP. En plus, nos recherches montrent que MyD88 est nécessaire pour l'expression de Smad4 et cela pour garantir une réponse optimale à travers la signalisation BMP6, conduisant ainsi à une expression adéquate de HAMP. Enfin, la protéine MyD88 joue un rôle crucial dans, la régulation de HAMP au niveau des macrophages, la diminution du taux du fer sérique en réponse au LPS et le maintien de l'homéostasie du fer.

Implicating the mechanisms of ADP-ribosylation factor activation in the resistance of invasive breast cancer cells to EGFR tyrosine kinase inhibitors

ADP-ribosylation factor-1 (ARF1) est une petite GTPase principalement connue pour son rôle dans la formation de vésicules au niveau de l’appareil de Golgi. Récemment, dans des cellules de cancer du sein, nous avons démontré qu’ARF1 est aussi un médiateur important de la signalisation du récepteur du facteur de croissance épidermique (EGFR) contrôlant la prolifération, la migration et l'invasion cellulaire. Cependant, le mécanisme par lequel l’EGFR active la GTPase ainsi que le rôle de cette dernière dans la régulation de la fonction du récepteur demeure inconnue. Dans cette thèse, nous avions comme objectifs de définir le mécanisme d'activation de ARF1 dans les cellules de cancer du sein hautement invasif et démontrer que l’activation de cette isoforme de ARF joue un rôle essentiel dans la résistance de ces cellules aux inhibiteurs de l'EGFR. Nos études démontrent que les protéines d’adaptatrices Grb2 et p66Shc jouent un rôle important dans l'activation de ARF1. Alors que Grb2 favorise le recrutement d’ARF1 à l'EGFR ainsi que l'activation de cette petite GTPase, p66Shc inhibe le recrutement du complexe Grb2-ARF1 au récepteur et donc contribue à limiter l’activation d’ARF1. De plus, nous démontrons que ARF1 favorise la résistance aux inhibiteurs des tyrosines kinases dans les cellules de cancer du sein hautement invasif. En effet, une diminution de l’expression de ARF1 a augmenté la sensibilité descellules aux inhibiteurs de l'EGFR. Nous montrons également que de hauts niveaux de ARF1 contribuent à la résistance des cellules à ces médicaments en améliorant la survie et les signaux prolifératifs à travers ERK1/2, Src et AKT, tout en bloquant les voies apoptotiques (p38MAPK et JNK). Enfin, nous mettons en évidence le rôle de la protéine ARF1 dans l’apoptose en réponse aux traitements des inhibiteurs de l’EGFR. Nos résultats indiquent que la dépletion d’ARF1 promeut la mort cellulaire induite par gefitinib, en augmentant l'expression de facteurs pro-apoptotiques (p66shc, Bax), en altérant le potentiel de la membrane mitochondriale et la libération du cytochrome C. Ensemble, nos résultats délimitent un nouveau mécanisme d'activation de ARF1 dans les cellules du cancer du sein hautement invasif et impliquent l’activité d’ARF1 comme un médiateur important de la résistance aux inhibiteurs EGFR.

Platelet adhesion signalling and the regulation of thrombus formation

Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) 1b-V-IX receptor complex, GPV1 and integrin alpha(2)beta(1)-These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin alphaIIbbeta(3), a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPV1 and integrins alpha(2)beta(1) and alpha(IIb)beta(3) contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis.

Lipoma preferred partner is a mechanosensitive protein regulated by nitric oxide in the heart

Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats (p < 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes (p < 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions (p < 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load.

The inositol-3-phosphate synthase biosynthetic enzyme has distinct catalytic and metabolic roles

Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in inositol auxotrophy that can only be partially rescued by exogenous inositol. Removal of inositol supplementation from the ino1- mutant results in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis and substrate adhesion. Inositol depletion also caused a dramatic generalised decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 independent of inositol biosynthesis. To characterise this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) was identified with homology to mammalian macromolecular complex adaptor proteins. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism.

Phenotypic modulation of cultured vascular smooth muscle cells: a functional analysis focusing on MLC and ERK1/2 phosphorylation

Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction-relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction-relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.

Genome-Wide Analysis of Differentially Expressed Genes During the Early Stages of Tomato Infection by a Potyvirus

Plant responses against pathogens cause up-and downward shifts in gene expression. To identify differentially expressed genes in a plant-virus interaction, susceptible tomato plants were inoculated with the potyvirus Pepper yellow mosaic virus (PepYMV) and a subtractive library was constructed from inoculated leaves at 72 h after inoculation. Several genes were identified as upregulated, including genes involved in plant defense responses (e. g., pathogenesis-related protein 5), regulation of the cell cycle (e. g., cytokinin-repressed proteins), signal transduction (e. g., CAX-interacting protein 4, SNF1 kinase), transcriptional regulators (e. g., WRKY and SCARECROW transcription factors), stress response proteins (e. g., Hsp90, DNA-J, 20S proteasome alpha subunit B, translationally controlled tumor protein), ubiquitins (e. g., polyubiquitin, ubiquitin activating enzyme 2), among others. Downregulated genes were also identified, which likewise display identity with genes involved in several metabolic pathways. Differential expression of selected genes was validated by macroarray analysis and quantitative real-time polymerase chain reaction. The possible roles played by some of these genes in the viral infection cycle are discussed.

Differential Evolutionary Constraints in the Evolution of Chemoreceptors: A Murine and Human Case Study

Chemoreception is among the most important sensory modalities in animals. Organisms use the ability to perceive chemical compounds in all major ecological activities. Recent studies have allowed the characterization of chemoreceptor gene families. These genes present strikingly high variability in copy numbers and pseudogenization degrees among different species, but the mechanisms underlying their evolution are not fully understood. We have analyzed the functional networks of these genes, their orthologs distribution, and performed phylogenetic analyses in order to investigate their evolutionary dynamics. We have modeled the chemosensory networks and compared the evolutionary constraints of their genes in Mus musculus, Homo sapiens, and Rattus norvegicus. We have observed significant differences regarding the constraints on the orthologous groups and network topologies of chemoreceptors and signal transduction machinery. Our findings suggest that chemosensory receptor genes are less constrained than their signal transducing machinery, resulting in greater receptor diversity and conservation of information processing pathways. More importantly, we have observed significant differences among the receptors themselves, suggesting that olfactory and bitter taste receptors are more conserved than vomeronasal receptors.

Docking, synthesis and pharmacological activity of novel urea-derivatives designed as p38 MAPK inhibitors

p38 mitogen-activated protein kinase (p38 MAPK) is an important signal transducing enzyme involved in many cellular regulations, including signaling pathways, pain and inflammation. Several p38 MAPK inhibitors have been developed as drug candidates to treatment of autoimmune disorders, such as rheumatoid arthritis. In this paper we reported the docking, synthesis and pharmacological activity of novel urea-derivatives (4a-e) designed as p38 MAPK inhibitors. These derivatives presented good theoretical affinity to the target p38 MAPK, standing out compound 4e (LASSBio-998), which showed a better score value compared to the prototype GK-00687. This compound was able to reduce in vitro TNF-alpha production and was orally active in a hypernociceptive murine model sensible to p38 MAPK inhibitors. Otherwise, compound 4e presented a dose-dependent analgesic effect in a model of antigen (mBSA)-induced arthritis and anti-inflammatory profile in carrageenan induced paw edema, indicating its potential as a new antiarthritis prototype. (c) 2012 Elsevier Masson SAS. All rights reserved.

Induktion von Pro-Autophagie-Signalen durch einen extra- oder intrazellulären Alpha-Toxin-Angriff

Autophagie ist ein konservierter, kataboler Mechanismus in allen eukaryoten Zellen. Unter anderem wird ihm eine wichtige Rolle als zellautonomer Abwehrmechanismus gegen Mikroorganismen zugeschrieben; von manchen Infektionserregern wird er jedoch unterlaufen oder sogar genutzt. Der stärkste Auslöser der Autophagie ist ein Mangel an Nährstoffen, insbesondere Aminosäuren. Über die Deaktivierung der Kinase mTORC1 und die Phosphorylierung des eukaryoten Translationsinitiationsfaktors eIF2α hemmt die Nährstoffknappheit die Proteinbiosynthese und aktiviert gleichzeitig Autophagie. Wie Mikroorganismen, insbesondere Bakterien, Autophagie auslösen oder manipulieren, ist derzeit Gegenstand intensiver Forschung. Modifikationen an Mikroben oder Phagosomen und Adapterproteine, die diese Veränderungen und Komponenten des Autophagieapparates erkennen, scheinen jedenfalls bei der selektiven Erkennung durch die Autophagie-Maschinerie wichtig zu sein. rnIn der vorliegenden Dissertationsarbeit wird die Rolle des membranporenbildenden α-Toxins von Staphylococcus aureus für die Induktion von Autophagie beleuchtet. Zum einen erwies sich die Akkumulation von (EGFP)-LC3(II), einem Marker der Autophagosomen, um intrazelluläre S. aureus als abhängig von α-Toxin. Zweitens, genügt extrazellulär appliziertes α-Toxin um (EGFP)-LC3(II)-positive Endosomen zu induzieren. Während der Angriff aus dem extrazellulären Raum jedoch binnen kurzer Zeit eine fokale Kumulation von phosphoryliertem eIF2α an der Plasmamembran induziert, die an der Internalisierung des Toxins beteiligt ist, findet sich am phagosomalen Kompartiment keine Toxin-abhängige Anhäufung von p-eIF2α oder proximalen Autophagieregulatoren. Dies impliziert, dass Toxin-Angriff auf die Plasmamembran, nicht aber auf das Phagosom, zu einer Reaktion führt, wie sie bei massivem Nährstoffmangel zu beobachten ist. Obwohl keine α-Toxin-abhängige Kumulation von p-eIF2α bei einem Angriff aus dem Phagosom erfolgt, findet sich um α-Toxin-produzierende Bakterien eine massive Kumulation von LC3 und Adapterprotein p62/Sequestosome1. Dies deutet daraufhin, dass der Ort des Angriffs - Plasmamembran oder Phagosom – für den Autophagie-induzierenden Mechanismus wichtig sein könnte. Der unterschiedliche Effekt auf die zellulären Ionenkonzentrationen, den ein Angriff auf die Plasmamembran oder auf ein Phagosom auslösen würde, bietet hierfür eine mögliche Erklärung. Die Aktivierung der Autophagie über Adapterproteine könnte dann als back-up Mechanismus fungieren, der auch dann greift, wenn eine Invasion ohne Schädigung der Plasmamembran erfolgt. Ein cross-talk der beiden Induktionswege ist angesichts der Bedeutung von p62 für die selektive und die Hunger-assoziierte Autophagie gut möglich; sezerniertes Toxin könnte durch die Aktivierung der basalen Autophagie Adapter-basierte Mechanismen verstärken.

Going against the tide--how encephalitogenic T cells breach the blood-brain barrier

During multiple sclerosis or its animal model, experimental autoimmune encephalomyelitis, circulating immune cells enter the central nervous system (CNS) causing neuroinflammation. Extravasation from the blood circulation across the vessel wall occurs through a multistep process regulated by adhesion and signal transducing molecules on the immune cells and on the endothelium. Since the CNS is shielded by the highly specialized blood-brain barrier (BBB), immune cell extravasation into the CNS requires breaching this particularly tight endothelial border. Consequently, travelling into the CNS demands unique adaptations which account for the extreme tightness of the BBB. Modern imaging tools have shown that after arresting on BBB endothelium, in vivo or in vitro encephalitogenic effector/memory T cells crawl for long distances, possibly exceeding 150 µm along the surface of the BBB endothelium before rapidly crossing the BBB. Interestingly, in addition to the distance of crawling, the preferred direction of crawling against the flow is unique for T cell crawling on the luminal surface of CNS microvessels. In this review, we will summarize the cellular and molecular mechanisms involved in the unique T cell behavior that is obviously required for finding a site permissive for diapedesis across the unique vascular bed of the BBB.

Abnormal collagen fibrils in cartilage of matrilin-1/matrilin-3-deficient mice

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.

Traffic Routes and Signals for the Tonoplast

Tonoplast, the membrane delimiting plant vacuoles, regulates ion, water and nutrient movement between the cytosol and the vacuolar lumen through the activity of its membrane proteins. Correct traffic of proteins from the endoplasmic reticulum (ER) to the tonoplast requires (i) approval by the ER quality control, (ii) motifs for exit from the ER and (iii) motifs that promote sorting to the tonoplast. Recent evidence suggests that this traffic follows different pathways that are protein-specific and could also reflect vacuole specialization for lytic or storage function. The routes can be distinguished based on their sensitivity to drugs such as brefeldin A and C834 as well as using mutant plants that are defective in adaptor proteins of vesicle coats, or dominant-negative mutants of Rab GTPases.

Nitric oxide induces cell death in canine cruciate ligament cells by activation of tyrosine kinase and reactive oxygen species

BACKGROUND: There is increasing evidence suggesting that development of progressive canine cranial cruciate ligament (CCL) rupture involves a gradual degeneration of the CCL itself, initiated by a combination of factors, ranging from mechanical to biochemical. To date, knowledge is lacking to what extent cruciate disease results from abnormal biomechanics on a normal ligament or contrary how far preliminary alterations of the ligament due to biochemical factors provoke abnormal biomechanics. This study is focused on nitric oxide (NO), one of the potential biochemical factors. The NO-donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in canine cranial and caudal cruciate ligament cells and to characterize signaling mechanisms during NO-stimulation. RESULTS: Sodium nitroprusside increased apoptotic cell death dose- and time-dependently in cruciate ligamentocytes. Cells from the CCL were more susceptible to apoptosis than CaCL cells. Caspase-3 processing in response to SNP was not detected. Testing major upstream and signal transducing pathways, NO-induced cruciate ligament cell death seemed to be mediated on different levels. Specific inhibition of tyrosine kinase significantly decreased SNP-induced cell death. Mitogen activated protein kinase ERK1 and 2 are activated upon NO and provide anti-apoptotic signals whereas p38 kinase and protein kinase C are not involved. Moreover, data showed that the inhibition reactive oxygen species (ROS) significantly reduced the level of cruciate ligament cell death. CONCLUSIONS: Our data support the hypothesis that canine cruciate ligamentocytes, independently from their origin (CCL or CaCL) follow crucial signaling pathways involved in NO-induced cell death. However, the difference on susceptibility upon NO-mediated apoptosis seems to be dependent on other pathways than on these tested in the present study. In both, CCL and CaCL, the activation of the tyrosine kinase and the generation of ROS reveal important signaling pathways. In perspective, new efforts to prevent the development and progression of cruciate disease may include strategies aimed at reducing ROS.

Cellular dynamic simulator: an event driven molecular simulation environment for cellular physiology.

In this paper, we present the Cellular Dynamic Simulator (CDS) for simulating diffusion and chemical reactions within crowded molecular environments. CDS is based on a novel event driven algorithm specifically designed for precise calculation of the timing of collisions, reactions and other events for each individual molecule in the environment. Generic mesh based compartments allow the creation / importation of very simple or detailed cellular structures that exist in a 3D environment. Multiple levels of compartments and static obstacles can be used to create a dense environment to mimic cellular boundaries and the intracellular space. The CDS algorithm takes into account volume exclusion and molecular crowding that may impact signaling cascades in small sub-cellular compartments such as dendritic spines. With the CDS, we can simulate simple enzyme reactions; aggregation, channel transport, as well as highly complicated chemical reaction networks of both freely diffusing and membrane bound multi-protein complexes. Components of the CDS are generally defined such that the simulator can be applied to a wide range of environments in terms of scale and level of detail. Through an initialization GUI, a simple simulation environment can be created and populated within minutes yet is powerful enough to design complex 3D cellular architecture. The initialization tool allows visual confirmation of the environment construction prior to execution by the simulator. This paper describes the CDS algorithm, design implementation, and provides an overview of the types of features available and the utility of those features are highlighted in demonstrations.