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New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
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BBA - Molecular Cell Research (v.1746, #2)
Potentials and pitfalls of DNA array analysis of the endothelial stress response by Dorothee Viemann; Klaus Schulze-Osthoff; Johannes Roth (pp. 73-84).
Endothelial cells respond to inflammatory stimuli with complex genetic alterations that determine the immune response and the outcome of the inflammatory process. An additional layer of complexity is added by the different phenotypes and functional heterogeneity of endothelial cells in the various tissues. To understand these complex gene response patterns and the regulatory pathways involved, many investigators increasingly use DNA microarray analysis. There are, however, many potential pitfalls in the use of microarrays that can result in false data and erroneous conclusions. This review surveys the principles of DNA microarray technology and its applications in endothelial cell research. We also attempt to outline some of the caveats and standard criteria that have to be considered in order to realize the full potential of microarrays in inflammation research.
Keywords: Endothelial cell; Gene expression; Inflammation; Microarray; Transcription
Pregnane and Xenobiotic Receptor (PXR/SXR) resides predominantly in the nuclear compartment of the interphase cell and associates with the condensed chromosomes during mitosis by Mallampati Saradhi; Aniruddha Sengupta; Gauranga Mukhopadhyay; Rakesh K. Tyagi (pp. 85-94).
Pregnane and Xenobiotic Receptor (PXR) is a transcription factor that is activated by a diverse range of xenobiotics and endogenous metabolites including steroids, bile acids and about 50% of the prescription drugs. In specific cell types (e.g. liver and intestine) it serves as a ‘xenosensor’ by regulating expression of a network of genes involved in xenobiotic clearance from the body. PXR expression in several cancerous tissues and its regulated expression of multi-drug resistance proteins highlight its significance in prognosis of malignancies. The view that subcellular localization and ligand induced movements of transcription factors is one of the major phenomena in regulating transcriptional activity, we used a green fluorescent protein tagged PXR chimera to study its dynamic behaviour in living cells. Under all experimental conditions, PXR was observed to be a predominantly nuclear protein maintaining a dynamic equilibrium between the nuclear and cytoplasmic compartments of the interphase cells. Interestingly, for the first time, a member of the nuclear receptor superfamily, PXR, has been observed to be associated with condensed chromosomes during all the mitotic stages of cell division. The significance of PXR association with mitotic chromosomes is discussed.
Keywords: Pregnane and Xenobiotic Receptor; Nuclear receptor; Cytochrome P-450; Chromosome condensation; Mitosis
Monte Carlo simulations of VEGF binding to cell surface receptors in vitro by Feilim Mac Gabhann; Michael T. Yang; Aleksander S. Popel (pp. 95-107).
The vascular endothelial growth factor (VEGF) family binds multiple endothelial cell surface receptors. Our goal is to build comprehensive models of these interactions for the purpose of simulating angiogenesis. In view of low concentrations of growth factors in vivo and in vitro, stochastic modeling of molecular interactions may be necessary. Here, we compare Monte Carlo simulations of the stochastic binding of VEGF and two of its major receptors on cells in vitro to equivalent deterministic simulations. In the range of typical VEGF concentrations, the stochastic and deterministic models are in agreement. However, we observe significant variability in receptor binding, which may be linked to biological stochastic events, e.g., blood vessel sprout initiation. We study patches of cell surface of varying sizes to investigate spatial integration of the signal by the cell, which impacts directly the variability of binding, and find significant variability up to the single-cell level. Dimerization of VEGF receptors does not significantly alter the variability in ligand binding. A ‘sliding window’ approach demonstrated no reduction in the variability of binding by temporal integration. The variability is expected to be more prominent in in vivo situations where the number of ligand molecules available for binding is less.
Keywords: Vascular endothelial growth factor; Angiogenesis; Stochastic model; Continuum approximation; Mathematical model; Ligand–receptor binding
Evidences for ubiquitination and intracellular trafficking of LAT, the linker of activated T cells by C. Brignatz; A. Restouin; G. Bonello; D. Olive; Y. Collette (pp. 108-115).
Current evidences indicate that T cells use protein sorting and degradation to control duration and specificity of T cell receptor (TcR) signalling, including the CD3ζ chain which is ubiquitinated upon TcR triggering. In a previous study, we showed that the Linker of activated T cells (LAT) is present at the plasma membrane and in transferrin-labelled intracellular compartments also containing the CD3ζ chain. Here we show that LAT protein levels are tightly regulated in Jurkat lymphoid T cells likely involving proteasome-dependent degradation, recycling through trans-Golgi/endosome compartments and clathrin-dependent internalisation. We further identify a novel post-translational modification of LAT by ubiquitination that is likely to influence LAT protein stability, intracellular localisation and/or recycling. Our results provide novel molecular and regulatory insights into the function of LAT adapter protein in T cell signalling.
Keywords: Linker of activated T cell; Ubiquitination; Adapter protein complexe; T cell signalling; Intracellular trafficking
Role of Pex19p in the targeting of PMP70 to peroxisome by Yoshinori Kashiwayama; Kota Asahina; Hiroyuki Shibata; Masashi Morita; Ania C. Muntau; Adelbert A. Roscher; Ronald J.A. Wanders; Nobuyuki Shimozawa; Masao Sakaguchi; Hiroaki Kato; Tsuneo Imanaka (pp. 116-128).
Pex19p is a protein required for the peroxisomal membrane synthesis. The 70-kDa peroxisomal membrane protein (PMP70) is synthesized on free cytosolic ribosomes and then inserted posttranslationally into peroxisomal membranes. Pex19p has been shown to play an important role in this process. Using an in vitro translation system, we investigated the role of Pex19p as a chaperone and identified the regions of PMP70 required for the interaction with Pex19p. When PMP70 was translated in the presence of purified Pex19p, a large part of PMP70 existed as soluble form and was co-immunoprecipitated with Pex19p. However, in the absence of Pex19p, PMP70 formed aggregates during translation. To identify the regions that interact with Pex19p, various truncated PMP70 were translated in the presence of Pex19p and subjected to co-immunoprecipitation. The interaction was markedly reduced by the deletion of the NH2-terminal 61 amino acids or the region around TMD6. Further, we expressed these deletion constructs of PMP70 in fusion with the green fluorescent protein in CHO cells. Fusion proteins lacking these Pex19p binding sites did not display any peroxisomal localization. These results suggest that Pex19p binds to PMP70 co-translationally and keeps PMP70 as a proper conformation for the localization to peroxisome.
Keywords: Peroxisome; Pex19p; Peroxisome membrane protein; ABC protein; PMP70
HIV infection is associated with increased NTPDase activity that correlates with CD39-positive lymphocytes by Daniela B.R. Leal; Cristiane A. Streher; Claudia de M. Bertoncheli; Luiz F.D. Carli; Claudio A.M. Leal; José E.P. da Silva; Vera M. Morsch; Maria R.C. Schetinger (pp. 129-134).
Infection with the human immunodeficiency virus (HIV) results in alterations in immune cells such as an increase or decrease of cytokine secretion and immunodeficiency. HIV causes a state of chronic cellular activation that can induce apoptosis in lymphocyte T-helpers, making the patient susceptive to opportunistic infections. The biochemical mechanisms involved in this immune response to HIV have been researched. Here, we have shown for the first time that ATP and ADP hydrolysis are essential for the immune response to HIV. Our results clearly indicate an increase of NTPDase-1 (EC 3.6.1.5) activity in lymphocytes of HIV-positive patients, confirmed by an enhanced CD39 expression on its surface. These results suggest that NTPDase-1 may be important to keep an adequate balance between the generation and consumption of ATP and to preserve cellular integrity and immune response to the HIV infection.
Keywords: NTPDase; CD39; Human lymphocyte; HIV-1
Oxidative modification of IκB by monochloramine inhibits tumor necrosis factor α-induced NF-κB activation by Tetsuya Ogino; Mutsumi Hosako; Kazuhisa Hiramatsu; Masako Omori; Michitaka Ozaki; Shigeru Okada (pp. 135-142).
We have previously reported that monochloramine (NH2Cl), a neutrophil-derived oxidant, inhibited tumor necrosis factor α (TNFα)-induced expression of cell adhesion molecules and nuclear factor-κB (NF-κB) activation (Free Radical Research 36 (2002) 845–852). Here, we studied the mechanism how NH2Cl inhibited TNFα-induced NF-κB activation, and compared the effects with taurine chloramine (Tau–NHCl). Pretreatment of Jurkat cells with NH2Cl at 70 μM resulted in suppression of TNFα-induced IκB phosphorylation and degradation, and inhibited NF-κB activation. In addition, a slow-moving IκB band appeared on SDS-PAGE. By contrast, Tau–NHCl for up to 200 μM had no effects. Interestingly, NH2Cl did not inhibit IκB kinase activation by TNFα. Protein phosphatase activity did not show apparent change. When recombinant IκB was oxidized by NH2Cl in vitro and phosphorylated by TNFα-stimulated Jurkat cell lysate, its phosphorylation occurred less effectively than non-oxidized IκB. In addition, when NF-κB–IκB complex was immunoprecipitated from NH2Cl-treated cells and phosphorylated in vitro by recombinant active IκB kinase, native IκB but not oxidized IκB was phosphorylated. Amino acid analysis of the in vitro oxidized IκB showed methionine oxidation to methionine sulfoxide. Although Tau–NHCl alone had little effects on TNFα-induced NF-κB activation, simultaneous presence of Tau–NHCl and ammonium ion significantly inhibited the NF-κB activation, probably through the conversion of Tau–NHCl to NH2Cl. These results indicated that NH2Cl inhibited TNFα-induced NF-κB activation through the oxidation of IκB, and that NH2Cl is physiologically more relevant than Tau–NHCl in modifying NF-κB-mediated cellular responses.
Keywords: Monochloramine; NF-κB; IκB; Methionine sulfoxide; TNFα; Inflammation
The Cas family docking protein, HEF1, promotes the formation of neurite-like membrane extensions by Sharmilla D. Bargon; Peter W. Gunning; Geraldine M. O'Neill (pp. 143-154).
The Cas family proteins are a family of adhesion docking molecules that mediate protein–protein interactions and contribute to a number of signal transduction pathways. Recent studies of two family members, p130Cas and Sin, have suggested that they may play a role in neurite formation. The current study demonstrates that the third family member, HEF1, can also stimulate the formation of neurite-like processes, in the presence of Rho kinase inhibitors. The HEF1-promoted processes actively extend from the cell body and resemble neurites both in the manner of process extension and in the distribution of adhesion-associated molecules. The HEF1-promoted processes are dependent on the presence of an intact microtubule system and can be inhibited by co-expression of either constitutively active Rac or Cdc42 GTPase. Together, our data support a role for the Cas proteins in regulating cellular morphologies that contribute to tissue specialization.
Keywords: Focal adhesion; Integrin; Cas protein; Neurite; Migration; GTPase
Fission yeast homologue of Tip41-like proteins regulates type 2A phosphatases and responses to nitrogen sources by Csaba Fenyvuesvolgyi; Robert T. Elder; Zsigmond Benko; Dong Liang; Richard Yuqi Zhao (pp. 155-162).
A fission yeast ( Schizosaccharomyces pombe) gene encoding a member of the TIP41-like protein family was identified and characterized. Deletion of the fission yeast tip41 gene leads to slower growth when ammonium chloride is the nitrogen source, but the growth rate is not affected when adenine is the nitrogen source. The tip41 mutant cells also enter the G1 phase of the cell cycle earlier than wild-type cells in response to nitrogen starvation. Overexpression of tip41+ causes cell death, spherical cell morphology and blocks the shift to G1 phase upon nitrogen starvation. Overexpression of tip41+ increases the activity of type 2A phosphatase. In a ppa2 deletion strain with reduced PP2A activity, overexpression of tip41+ no longer blocks the shift to G1 upon nitrogen starvation. These results suggest that fission yeast Tip41 plays a role in cellular responses to nitrogen nutrient conditions at least partly through regulation of type 2A phosphatase activity.
Keywords: Schizosaccharomyces pomb; e; Tip41-like family protein; Nitrogen starvation; PP2A
Impact of modeled microgravity on microvascular endothelial cells by Sabrina Cotrupi; Daniela Ranzani; Jeanette A.M. Maier (pp. 163-168).
Microvascular endothelial cells are protagonists in inflammation and angiogenesis. They contribute to the integrity of microvasculature by synthesizing a large array of cytokines, growth factors and mediators active on the endothelium itself, on smooth muscle cells and circulating leukocytes. Because space flight (i) associates with vascular impairment and (ii) modulates the cytokine network, we evaluated the effect of modeled microgravity on microvascular 1G11 cells. We found that modeled microgravity reversibly inhibits endothelial growth and this correlates with an upregulation of p21, a cyclin-dependent kinases inhibitor. By protein array, we found that microgravity inhibits the synthesis of interleukin 6, an event that may contribute to growth retardation. We also detected increased amounts of nitric oxide, a mediator of inflammatory responses, a potent vasodilator and a player in angiogenesis. The increased synthesis of nitric oxide is due, at least in part, to an upregulation of endothelial nitric oxide synthase. Because low levels of IL-6 might contribute to endothelial growth retardation as well as to the enhancement of nitric oxide synthesis, we hypothesize a central role of IL-6 in modulating microvascular endothelial cell behaviour in modeled microgravity.
Keywords: Endothelial cell; Microvasculature; Cytokine; Nitric oxide; Microgravity