PAF and the role of the vagus nerve in the breathing pattern of the pig.

In 14 anesthetized, spontaneously breathing pigs we examined the changes in breathing pattern, in respiratory mechanics and in systemic and pulmonary vascular parameters after i.v. PAF administration. In another 3 pigs, the effects of PAF were also examined after bilateral vagotomy. In intact pigs, PAF induces apnea, bronchoconstriction, pulmonary hypertension and systemic hypotension. Our results also show that administration of PAF alters the phasic vagal activity, modifying the slope of VT vs TI and TE vs TI relationships and the TI0/TI ratio. These effects and apnea are vagus-dependent. The central excitatory timing effect of PAF on inspiratory duration (TI0) was correlated with a decrease in passive compliance but not with active compliance. We postulate that the activation by vagal input strengthens the mechanisms that counteract the bronchoconstrictor effect of PAF.     

Genes encoding a histone H3.3-like variant in Arabidopsis contain intervening sequences.

Two genes encoding a particular H3 histone variant were isolated from Arabidopsis thaliana. These genes differ from the H3 genes previously cloned from Arabidopsis and other plants by several interesting properties: (1) the two genes are located close to each other; (2) their coding regions are interrupted by two or three small introns, the two closest to the initiation codon being located at the same place in the two genes; (3) another, long intron is located in the 5'-untranslated region just before the initiation codon of gene I as deduced from the sequence of several corresponding cDNAs, and very likely also of gene II; (4) these genes do not show preferential expression in organs containing meristematic tissues contrary to the classical intronless replication-dependent histone genes, thus suggesting that their expression is not replication-dependent; (5) the protein encoded by both genes is the same and corresponds to a minor H3 variant highly conserved among all the plant species studied up to now. All these characteristics are common with the animal replication-independent H3.3 histone genes and it is assumed that the genes described here are the first example of the equivalent H3.3 gene family in plants. Interestingly, the promoter regions of the two genes have the same general structure as the Arabidopsis intronless genes. Possible implications on the regulation of H3 genes expression are discussed.     

Distinct patterns of insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 expression in oxidant exposed lung epithelial cells

Oxygen (O(2)) species are involved in a large variety of pulmonary diseases. Among the various cell types that compose the lung, the epithelial cells of the alveolar structure appear to be a major target for oxidant injury. Despite their importance in the repair processes, the mechanisms which regulate the replication of the stem cells of the alveolar epithelium, the type 2 cells, remain poorly understood. Based on the results of several studies which have documented the involvement of the insulin-like growth factor (IGF) system in lung epithelial cell replication, and which have also suggested a role for IGF binding proteins (IGFBPs) in the control of cell proliferation, the aim of the present work was to determine whether IGFBPs could be involved in the modulation of growth of human lung epithelial cells exposed to oxidants. Experiments were performed using a human lung adenocarcinoma cell line (A549) which was exposed for various durations to hyperoxia (95% O(2)). We observed a rapid and reversible growth arrest of the cells after only 24 h of O(2) exposure. When oxidant injury was prolonged, growth arrest was followed by induction of apoptosis with activation of the Fas pathway. These effects were associated with an increased expression of IGFBP-2 and IGFBP-3. In addition, study of localization of these proteins revealed distinct patterns of distribution. IGFBP-3 was mainly present in the extracellular compartment. In comparison, the fraction of IGFBP-2 secreted was less abundant whereas the IGFBP-2 fraction in the intracellular compartment appeared stronger. In addition, analysis of the subcellular localization provided data indicating the presence of IGFBP-2 in the nucleus. Taken together these data support a role for IGFBP-2 and IGFBP-3 in the processes of growth arrest and apoptosis in lung epithelial cells upon oxidant exposure. They also suggest that distinct mechanisms may link IGFBP-2 and IGFBP-3 to the key regulators of the cell cycle.     

Molecular organization and dynamics of the melatonin MT1 receptor/RGS20/Gi protein complex reveal asymmetry of receptor dimers for RGS and Gi coupling

Functional asymmetry of G‐protein‐coupled receptor (GPCR) dimers has been reported for an increasing number of cases, but the molecular architecture of signalling units associated to these dimers remains unclear. Here, we characterized the molecular complex of the melatonin MT₁ receptor, which directly and constitutively couples to G_i proteins and the regulator of G‐protein signalling (RGS) 20. The molecular organization of the ternary MT₁/G_i/RGS20 complex was monitored in its basal and activated state by bioluminescence resonance energy transfer between probes inserted at multiple sites of the complex. On the basis of the reported crystal structures of G_i and the RGS domain, we propose a model wherein one G_i and one RGS20 protein bind to separate protomers of MT₁ dimers in a pre‐associated complex that rearranges upon agonist activation. This model was further validated with MT₁/MT₂ heterodimers. Collectively, our data extend the concept of asymmetry within GPCR dimers, reinforce the notion of receptor specificity for RGS proteins and highlight the advantage of GPCRs organized as dimers in which each protomer fulfils its specific task by binding to different GPCR‐interacting proteins.     

Obesity and melanoma: could fat be fueling malignancy?

Over the last decade, it has become increasingly clear that adipose tissue, and particularly adipocytes, contributes to tumor progression. Obesity, an ever‐increasing worldwide phenomenon, exacerbates this effect. The influence of obesity on melanoma remains poorly studied, although recent data do underline an association between the two diseases in both humans and murine models. Herein, we review the impact of obesity on melanoma incidence and progression and discuss the underlying mechanisms known to be involved. Adipose tissue favors the proliferation and aggressiveness of melanoma cells through a direct dialog, mediated by soluble factors and by exosomes, and through remodeling of the tumor microenvironment. This knowledge could, in the future, help to design new personalized therapeutic options for obese melanoma patients.     

An unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies

mRNA deadenylation is a key process in the regulation of translation and mRNA turnover. In Saccharomyces cerevisiae, deadenylation is primarily carried out by the Ccr4p and Caf1p/Pop2p subunits of the Ccr4-Not complex, which is conserved in eukaryotes including humans. Here we have identified an unconventional human Ccr4-Caf1 complex containing hCcr4d and hCaf1z, distant human homologs of yeast Ccr4p and Caf1p/Pop2p, respectively. The hCcr4d-hCaf1z complex differs from conventional Ccr4-Not deadenylase complexes, because (i) hCaf1z and hCcr4d concentrate in nuclear Cajal bodies and shuttle between the nucleus and cytoplasm and (ii) the hCaf1z subunit, in addition to rapid deadenylation, subjects substrate RNAs to slow exonucleolytic degradation from the 3' end in vitro. Exogenously expressed hCaf1z shows both of those activities on reporter mRNAs in human HeLa cells and stimulates general mRNA decay when restricted to the cytoplasm by deletion of its nuclear localization signal. These observations suggest that the hCcr4d-hCaf1z complex may function either in the nucleus or in the cytoplasm after its nuclear export, to degrade polyadenylated RNAs, such as mRNAs, pre-mRNAs, or those RNAs that are polyadenylated prior to their degradation in the nucleus.     

Genome-wide gene expression profiling of microgravity effect on human liver cells

Human exposure to microgravity is considered the major environmental factor of space flight that affects cells and tissues causing adverse effects to human health. Ground-based gravity-simulation experiments at the cellular and molecular levels have gained some insight into the underlying molecular and cellular alterations induced by microgravity. However, systematic study and detailed molecular mechanisms of the adverse effect of microgravity on living cells are still lacking. The main objective of this study was to apply DNA microarray technology in time-course experiments for genome-wide search of genes whose expression are altered by microgravity, as part of the effort in the identification of major space genes. In this study, we analyzed global gene expression profiles for a human liver cell line exposed to a ground-based modeled microgravity system for 1, 3, and 4 days using the rotary cell culture system (RCCS) and the Agilent 22k human oligo DNA microarrays. We have found that 139 genes' mRNA levels were significantly (P < or = 0.01) altered by the microgravity exposures. Some of these identified genes were further verified by Northern analysis.     

Hydrogen peroxide. Ubiquitous in cell culture and in vivo?

Hydrogen peroxide (H2O2) is widely regarded as a cytotoxic agent whose levels must be minimized by the action of antioxidant defence enzymes. In fact, H2O2 is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H2O2 may be greater than is commonly supposed; levels of H2O2 in the human body may be controlled not only by catabolism but also by excretion, and H2O2 could play a role in the regulation of renal function and as an antibacterial agent in the urine. Cell culture is a widely used method for the investigation of "physiological" processes such as signal transduction and regulation of gene expression, but chemical reactions involving cell culture media are rarely considered. Addition of reducing agents to commonly used cell-culture media can lead to generation of substantial amounts of H2O2. Some or all of the reported effects of ascorbic acid and polyphenolic compounds (e.g., quercetin, catechin, epigallocatechin, epigallocatechin gallate) on cells in culture may be due to H2O2 generation by interaction of these compounds with cell culture media.     

Cell cycle-dependent phosphorylation and ubiquitination of a G protein alpha subunit

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G(1) phase. Here, we show that the Gα subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G(2)/M. Accordingly, phosphorylation of Gpa1 in G(2)/M phase leads to polyubiquitination in G(1) phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.