Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state-specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways

Human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. In the present study, we analyzed the roles of focal adhesion kinase (Fak)/Src signaling to the PI3-K/Akt-1 and mitogen-activated protein kinase (MEK)/extracellular regulated kinases (Erk) pathways, within the context of such differentiation-state distinctions. Anoikis was induced by inhibition of beta1 integrins (antibody blocking), inhibition of Fak (pharmacologic inhibition or overexpression of dominant negative mutants), or by maintaining cells in suspension. Activation parameters of Fak, Src, Akt-1, and Erk1/2 were analyzed. Activities of Src, Akt-1, or Erk1/2 were also blocked by pharmacological inhibition or by overexpression of dominant-negative mutants. We report that: (1) the loss or inhibition of beta1 integrin binding activity causes anoikis and results in a down-activation of Fak, Src, Akt-1, and Erk1/2 in both undifferentiated, and differentiated cells; (2) the inhibition of Fak likewise causes anoikis and a down-activation of Src, Akt-1, and Erk1/2, regardless of the differentiation state; (3) Src, PI3-K/Akt-1, and MEK/Erk contribute to the survival of differentiated cells, whereas MEK/Erk does not play a role in the survival of undifferentiated ones; (4) the inhibition/loss of beta1 integrin binding and/or Fak activity results in a loss of Src engagement with Fak, regardless of the state of differentiation; and (5) Src contributes to the activation of both the PI3-K/Akt-1 and MEK/Erk pathways in undifferentiated cells, but does not influence PI3-K/Akt-1 in differentiated ones. Hence, Fak/Src signaling to the PI3-K/Akt-1 and MEK/Erk pathways undergoes a differentiation state-specific uncoupling which ultimately reflects upon the selective engagement of these same pathways in the mediation of intestinal epithelial cell survival.     

Colonic CD90+ Crypt Fibroblasts Secrete Semaphorins to Support Epithelial Growth

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Role of the mod(mdg4) common region in homolog segregation in Drosophila male meiosis

Homologous chromosomes must pair and establish stable connections during prophase I of meiosis to segregate reliably from each other at anaphase I. In most organisms, the stable connections, called chiasmata, arise from crossovers. In Drosophila males, homologs pair and segregate without crossing over. Chiasmata are replaced by a homolog conjunction complex that includes the Stromalin in Meiosis (SNM) and Modifier of Mdg4 in Meiosis (MNM) proteins. MNM is one of 31 alternative splice products of mod(mdg4), all of which share a common 402-amino-acid N terminus and differ at their C termini. Previous data demonstrated that an MNM-specific exon is required for homolog conjunction, but did not address whether the N-terminal common region, which includes a BTB domain that can mediate coalescence of protein-DNA complexes, is also required. Here we describe a mutation in the common region of mod(mdg4), Z3-3401, that causes qualitatively similar phenotypes as the MNM-specific alleles but disrupts X-Y segregation much more drastically than autosomal segregation. The mutant MNM protein in Z3-3401 is expressed throughout prophase I in spermatocytes but the protein is confined to the cytoplasm, suggesting that the Z3-3401 mutation disrupts a signal required for nuclear localization or retention. Z3-3401 fails to complement a large battery of lethal and semilethal alleles in the common region for meiotic nondisjunction, including an allele containing an amino acid substitution at a conserved residue in the BTB/POZ domain, consistent with a general requirement for the mod(mdg4) common region in homolog segregation.     

Synapsis-defective mutants reveal a correlation between chromosome conformation and the mode of double-strand break repair during Caenorhabditis elegans meiosis

SYP-3 is a new structural component of the synaptonemal complex (SC) required for the regulation of chromosome synapsis. Both chromosome morphogenesis and nuclear organization are altered throughout the germlines of syp-3 mutants. Here, our analysis of syp-3 mutants provides insights into the relationship between chromosome conformation and the repair of meiotic double-strand breaks (DSBs). Although crossover recombination is severely reduced in syp-3 mutants, the production of viable offspring accompanied by the disappearance of RAD-51 foci suggests that DSBs are being repaired in these synapsis-defective mutants. Our studies indicate that once interhomolog recombination is impaired, both intersister recombination and nonhomologous end-joining pathways may contribute to repair during germline meiosis. Moreover, our studies suggest that the conformation of chromosomes may influence the mode of DSB repair employed during meiosis.     

Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line

The replication of chromosomes in meiosis is an important first step for subsequent chromosomal interactions that promote accurate disjunction in the first of two segregation events to generate haploid gametes. We have developed an assay to monitor DNA replication in vivo in mitotic and meiotic germline nuclei of the nematode Caenorhabditis elegans. Using mutants that affect the mitosis/meiosis switch, we show that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cell nuclei. Furthermore, our assay reveals that different regions of the genome replicate at different times, with the heterochromatic-like X chromosomes replicating at a distinct time from the autosomes. Finally, we have exploited S-phase labeling to monitor the timing of progression through meiotic prophase. Meiotic prophase for oocyte production in hermaphrodites lasts 54-60 h. Further, we find that the duration of the pachytene sub-stage is modulated by the presence of sperm. On the other hand, meiotic prophase for sperm production in males is completed by 20-24 h. Possible sources for the sex-specific differences in meiotic prophase kinetics are discussed.     

Lipases in lipophilization reactions

Lipases are used in various sectors, as pharmaceutical, food or detergency industry. Their advantage versus classical chemical catalysts is that they exhibit a better selectivity and operate in milder reaction conditions. Theses enzymes can also be used in lipophilization reactions corresponding to the grafting of a lipophilic moiety to a hydrophilic one such as sugar, amino acids and proteins, or phenolic compounds. The major difficulty to overcome in such enzyme-catalyzed reaction resides in the fact that the two involved substrates greatly differ in term of polarity and solvent affinity. Therefore, several key parameters are to be considered in order to achieve the reaction in satisfactory kinetics and yields. The present review discusses the nature of such parameters (eg solvent nature, water activity, chemical modification of substrates) and illustrates their effect with examples of lipase-catalyzed lipophilization reactions of various sugar, amino acids or phenolic derivatives.     

Leukotriene B4 Enhances NOD2-Dependent Innate Response against Influenza Virus Infection

Leukotriene B₄ (LTB₄), a central mediator of inflammation, is well known for its chemoattractant properties on effectors cells of the immune system. LTB₄ also has the ability to control microbial infection by improving host innate defenses through the release of antimicrobial peptides and modulation of intracellular Toll-like receptors (TLRs) expression in response to agonist challenge. In this report, we provide evidences that LTB₄ acts on nucleotide-binging oligomerization domain 2 (NOD2) pathway to enhance immune response against influenza A infection. Infected mice receiving LTB₄ show improved survival, lung architecture and reduced lung viral loads as compared to placebo-treated animals. NOD2 and its downstream adaptor protein IPS–1 have been found to be essential for LTB₄-mediated effects against IAV infection, as absence of NOD2 or IPS–1 diminished its capacity to control viral infection. Treatment of IAV-infected mice with LTB₄ induces an increased activation of IPS-1-IRF3 axis leading to an enhanced production of IFNβ in lungs of infected mice. LTB₄ also has the ability to act on the RICK-NF-κB axis since administration of LTB₄ to mice challenged with MDP markedly increases the secretion of IL–6 and TNFα in lungs of mice. TAK1 appears to be essential to the action of LTB₄ on NOD2 pathway since pretreatment of MEFs with TAK1 inhibitor prior stimulation with IAV or MDP strongly abrogated the potentiating effects of LTB₄ on both IFNβ and cytokine secretion. Together, our results demonstrate that LTB₄, through its ability to activate TAK1, potentiates both IPS–1 and RICK axis of the NOD2 pathway to improve host innate responses.     

Visualizing Compound Distribution during Zebrafish Embryo Development: The Effects of Lipophilicity and DMSO

The predictability of the zebrafish embryo model is highly influenced by internal exposure of the embryo/larva. As compound uptake is likely to be influenced by factors such as lipophilicity, solvent use, and chorion presence, this article focuses on investigating their effects on compound distribution within the zebrafish embryo. To visualize compound uptake and distribution, zebrafish embryos were exposed for 96 hr, starting at 4 hr postfertilization, to water‐soluble dyes: Schiff's reagent (logP –4.63), Giemsa stain (logP –0.77), Van Gierson stain (logP 1.64), Cresyl fast violet (logP 3.5), Eosine Y (logP 4.8), Sudan III (logP 7.5), and Oil red O (logP 9.81), with and without 1% dimethyl‐sulfoxide (DMSO). Three additional compounds were used to analytically determine the uptake and distribution: Acyclovir (logP –1.56), Zidovudine (logP 0.05), and Metoprolol Tartrate Salt (logP 1.8). Examinations were performed every 24 hr. Both methods (visualization and specific analysis) showed that exposure to higher logP values results in higher compound uptake. Specific analysis showed that for lipophilic compounds >90% of compound is taken up by the embryo. For hydrophilic compounds, >90% of compound within the complete egg could not be associated to embryo or chorion and is probably distributed into the perivitelline space. Overall, internal exposure analyses on at least two occasions (i.e., before and after hatching) is crucial for interpretation of zebrafish embryotoxicity data, especially for compounds with extreme logP values. DMSO did not affect exposure when examined with the visualization method, however, this method might be not sensitive enough to draw hard conclusions.