lca_algebraic: a library bringing symbolic calculus to LCA for comprehensive sensitivity analysis

The International Journal of Life Cycle Assessment - In this paper, we present new tools to ease the analysis of the effect of variability and uncertainty on life cycle assessment (LCA) results....     

Differential role of lipid rafts in the functions of CD4+ and CD8+ human T lymphocytes with aging

Lipid rafts are critical to the assembly of the T-cell receptor (TCR) signaling machinery. It is not known whether lipid raft properties differ in CD4+ and CD8+ T cells and whether there are age-related differences that may account in part for immune senescence. Data presented here showed that time-dependent interleukin-2 (IL-2) production was different between CD4+ and CD8+ T cells. The defect in IL-2 production by CD4+ T cells was not due to lower levels of expression of the TCR or CD28. There was a direct correlation between the activation of p56(Lck) and LAT and their association/recruitment with the lipid raft fractions of CD4+ and CD8+ T cells. p56Lck, LAT and Akt/PKB were weakly phosphorylated in lipid rafts of stimulated CD4+ T cells of elderly as compared to young donors. Lipid rafts undergo changes in their lipid composition (ganglioside M1, cholesterol) in CD4+ and CD8+ T cells of elderly individuals. This study emphasizes the differential role of lipid rafts in CD4+ and CD8+ T-cell activation in aging and suggests that the differential localization of CD28 may explain disparities in response to stimulation in human aging.     

Activation of MAP kinases in growth responsive pancreatic cancer cells.

The implication of MAP kinases in the proliferation control of pancreatic cancer cells is still unknown. This study was undertaken to examine the contribution of the p44/p42 and p38 MAP kinases in the mitogenic response to epidermal growth factor (EGF) and bombesin in human pancreatic cancer cells, MIA PaCa-2 and PANC-1. Data indicate that EGF and bombesin stimulated growth of both cell lines. In MIA PaCa-2 cells, EGF and bombesin stimulated the in gel activation of p38 while p44/p42 kinases exhibited high basal activity and no response to stimuli. Growth and p38 activation were inhibited by genistein, wortmannin, PD98059 and SB203580, specific inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, MEK-1 and p38 kinases, respectively. In PANC-1 cells, EGF and bombesin stimulated p42 in gel activation; p44 remained highly activated and unresponsive to stimuli and p38 did not respond. Stimulated growth and p42 activation were inhibited by genistein, wortmannin and PD98059. Estimation of MAPK activities with a specific anti-active MAP kinase antibody indicated, however, that EGF increased the intensity of the bands corresponding to p42 and p44 MAP kinases in both cell lines, indicating that the mitogenic factor can regulate MAP kinase activity. Data also pointed out that ATP is sufficient to increase MAP kinase activity within the in gel assay technique and may thus explain the discrepancies existing between the in gel assay data and those obtained with the anti-active MAP kinase antibody.     

Bimodal regulation of RAF by CNK in Drosophila

Connector enhancer of KSR (CNK) is a multidomain-containing protein previously identified as a positive regulator of the RAS/MAPK pathway in DROSOPHILA: Using transfection experiments and an RNAi-based rescue assay in Drosophila S2 cells, we demonstrate that CNK has antagonistic properties with respect to RAF activity. We show that CNK's N-terminal region contains two domains (SAM and CRIC) that are essential for RAF function. Unexpectedly, we also report that the C-terminal region of CNK contains a short bipartite element that strongly inhibits RAF catalytic function. Interestingly, CNK's opposite properties appear to prevent signaling leakage from RAF to MEK in the absence of upstream signals, but then transforms into a potent RAF activator upon signal activation. Together, these findings suggest that CNK not only participates in the elusive RAF activation process, but might also contribute to the switch-like behavior of the MAPK module.     

Src42 binding activity regulates Drosophila RAF by a novel CNK-dependent derepression mechanism

Connector enhancer of KSR (CNK), an essential component of Drosophila receptor tyrosine kinase/mitogen-activated protein kinase pathways, regulates oppositely RAF function. This bimodal property depends on the N-terminal region of CNK, which integrates RAS activity to stimulate RAF and a bipartite element, called the RAF-inhibitory region (RIR), which binds and inhibits RAF catalytic activity. Here, we show that the repressive effect of the RIR is counteracted by the ability of Src42 to associate, in an RTK-dependent manner, with a conserved region located immediately C-terminal to the RIR. Strikingly, we found that several cnk loss-of-function alleles have mutations clustered in this area and provide evidence that these mutations impair Src42 binding. Surprisingly, the derepressing effect of Src42 does not appear to involve its catalytic function, but critically depends on the ability of its SH3 and SH2 domains to associate with CNK. Together, these findings suggest that the integration of RTK-induced RAS and Src42 signals by CNK as a two-component input is essential for RAF activation in Drosophila.     

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2–7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.DNA replication during the S phase necessitates that the entire genome be duplicated with the minimum of errors. Thousands of replication forks are involved in this process and they must be coordinated to ensure that every section of DNA is only replicated once. Errors in DNA replication are likely to be a major cause of the genetic instability that can lead to cancer (1). Cells are able to prevent duplicate replication of DNA by having a distinct stage that occurs during the G1 phase when replication origins are “licensed” for replication, a process that involves the preloading of several proteins involved in DNA replication (2). As DNA is replicated at each origin, these proteins are removed, thereby ensuring that each origin fires only once during each S phase. DNA damage response kinases activated by the stalled forks prevent the replication machinery from being activated in new chromosome domains, indicating a tight relationship between the DNA damage response and the DNA replication pathways (3, 4).The first step of the replication licensing mechanism is the loading of the minichromosome maintenance (MCM)¹ proteins on to replication origins along with origin recognition complex proteins, Cdt6 and Cdt1 (5). The eukaryotic MCM complex consists of six paralogs that form a heterohexameric ring. All eukaryotic organisms possess six homologous proteins (MCM2-MCM7) that form a heterohexameric ring that belong to the family of AAA+ (ATPase associated with various cellular activities) proteins and share similarities to other hexameric helicases (6). Even though additional MCM proteins have been identified in higher eukaryotes, the MCM2-MCM7 complex remains the prime candidate for the role of replicative helicase (7). MCM2–7 is required for both initiation and elongation of DNA replication, with its regulation at each stage being an essential player of eukaryotic DNA replication (8). As a critical mechanism to ensure only a single round of DNA replication, the loading of additional MCM2–7 complexes onto origins of replication is inactivated by redundant mechanisms after passage into S phase (9).The MCM complex plays a crucial role in determining the replication potential of cells, but recent work suggests that MCM proteins are not only targets of the S-phase checkpoints, but they also interact directly with components of the checkpoint and repair pathways (10, 11). In yeast, temperature sensitive MCM cells at restrictive temperature contain numerous foci recognized by the phosphorylated histone H2AX antibody (12), suggesting a role in the repair of DNA double-strand breaks. Although, in principle, only two DNA helicase activities are required to establish a bidirectional replication fork from each origin, a relatively large excess of MCM complexes are loaded at origins of replication and distributed along the chromatin (13). Their function is not well understood, and most of them are displaced from the DNA during S-phase, apparently without having played an active role in DNA replication. The “MCM paradox” refers to the fact that, at least in yeast, Xenopus, Drosophila, and mammalian cells, it is possible to reduce the concentration of MCM proteins by more than 90% without impairing DNA replication (14–18) and also refers to the observation that the majority of MCM complexes do not localize to the sites of DNA synthesis in mammalian cells, further suggesting a potential role for the MCM proteins beyond DNA replication.Using a combination of stable isotope labeling with amino acids in cell culture (SILAC)–based quantitative proteomics (19) with immunoprecipitation of green fluorescent protein (GFP)-tagged fusion proteins (20), we identified differences in protein binding partners with the MCM complex following DNA damage. Stable cell lines expressing GFP-tagged MCM2 and MCM5 were used in immunoprecipitation experiments from cells that were either mock treated, or treated with Etoposide for 15, 60, and 240 min. Etoposide is an antitumor drug that stabilizes a covalent complex between the DNA topoisomerase II and DNA by interfering with the cleavage-ligation reaction of the topoisomerase (21). This revealed specific interaction between the MCM complex and several proteins such as Nucleophosmin, BAG2, UPP1, and HDAC10. Interestingly, the MCM complex showed dynamic changes in interaction with Importin7 and the histone chaperone ASF1, and a decrease in interaction with the Chromodomain helicase DNA binding protein 3 (CHD3) resulting from the treatment with etoposide. This increase in interaction with ASF1 was followed by an enrichment of histone proteins, suggesting a novel role for the MCM proteins in histone deposition on chromatin following DNA damage.     

The role of the MAPK pathway alterations in GM-CSF modulated human neutrophil apoptosis with aging

Background  

Neutrophils represent the first line of defence against aggressions. The programmed death of neutrophils is delayed by pro-inflammatory stimuli to ensure a proper resolution of the inflammation in time and place. The pro-inflammatory stimuli include granulocyte-macrophage colony-stimulating factor (GM-CSF). Recently, we have demonstrated that although neutrophils have an identical spontaneous apoptosis in elderly subjects compared to that in young subjects, the GM-CSF-induced delayed apoptosis is markedly diminished. The present study investigates whether an alteration of the GM-CSF stimulation of MAPKs play a role in the diminished rescue from apoptosis of PMN of elderly subjects.     

Signal transduction and functional changes in neutrophils with aging

It is well known that the immune response decreases during aging, leading to a higher susceptibility to infections, cancers and autoimmune disorders. Most widely studied have been alterations in the adaptive immune response. Recently, the role of the innate immune response as a first-line defence against bacterial invasion and as a modulator of the adaptive immune response has become more widely recognized. One of the most important cell components of the innate response is neutrophils and it is therefore important to elucidate their function during aging. With aging there is an alteration of the receptor-driven functions of human neutrophils, such as superoxide anion production, chemotaxis and apoptosis. One of the alterations underlying these functional changes is a decrease in signalling elicited by specific receptors. Alterations were also found in the neutrophil membrane lipid rafts. These alterations in neutrophil functions and signal transduction that occur during aging might contribute to the significant increase in infections in old age.