Isolation of oligomycin-sensitive adenosine triphosphatase from beef heart mitochondria and analysis of its fine structure

1. An oligomycin -sensitive ATPase was isolated and partially purified from beef heart mitochondria. The specific activity of ATPase sensitive to oligomycin of the fraction was five to eight times that of aged mitochondrial or of DNP-induced mitochondrial ATPase assayed under the same condition. 2. Electron micrographs of the partially purified oligomycin- sensitive ATPase reveal a structure in which headpieces are regularly attached by way of stalks to a thread-like structure derived from a superficial portion of base pieces. 3. A high concentration of the structured material coincided with a high activity of oligomycin-sensitive ATPase. When the headpieces were detached from the structure, the ATPase became insensitive to oligomycin. 4. The fraction of oligomycin -sensitive ATPase was essentially free of membrane structure and was contaminated with a small amount of cytochromes b and Cl but no cyt. a. Cytochrome concentrations of the preparations were indifferent to the activity of oligomycin sensitive ATPase. It follows that ATPase does not require cytochromes or membrane structure for its oligomycin sensitivity. 5. From these results it seems that the factor rendering ATPase sensitive to oligomycin should be contained in the stalks and/or the thread-like portion of basepieces of the structure. The structure is the simplest unit of oligomycinsensitive ATPase as yet obtained. 6. The structure was called &#34;oligomycin-sensitive ATPase particles&#34; (abbreviated as OSA particles). A unit of OSA particles consists of a headpiece attached by a stalk to a portion of base piece.</p

Horizontal gene transfer contributed to the evolution of extracellular surface structures

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment

A PPAR-gamma, egy lipidek által aktivált transzkripciós faktor aktivitásának szabályozása a makrofágok különböző állapotaiban = Regulation of the activity of a lipid-activated transcription factor, PPAR-gamma in macrophages

A Peroxisome Proliferator-activated Receptor y (PPARy) egy lipidek által aktivált transzkripciós faktor, mely a lipidanyagcsere és a gyulladás szabályozásával olyan folyamatokban vesz részt, mint az érelmeszesedés és diabétesz. A PPARy aktivátorai több szinten képesek a gyulladás gátlására. Jelen pályázat célja az volt, hogy tanulmányozzuk a gyulladásos folyamatok PPARy aktivitására kifejtett hatását, megvizsgáljuk, hogy a gyulladásos mediátorok hogyan kommunikálnak a PPARy-val és megfejtsük ezeknek folyamatoknak a molekuláris mechanizmusát. Azt találtuk, hogy a gyulladásos molekulák befolyásolják a PPARy működését. Proinflammatórikus molekulák gátolják, míg az interleukin-4 (IL-4) fokozza a receptor működését makrofágokban és dendritikus sejtekben. Az IL-4 szignálútvonal bekapcsolása egy újonnan feltárt mechanizmussal, a Signal Transducer and Activators of Transcription 6 (STAT6) és a PPARg interakciója révén erősíti a receptor válaszait a célgének promóterén. Ennek eredményeképpen az IL-4 emeli a PPARg által szabályozott gének számát és fokozza az egyes gének esetében azok transzkripcióját. Ily módon a PPARg egy pozitív transzkripciós faktorként működhet a makrofágokban is. Eredményeink szerint létezik egy új mechanizmus, ahogy az immunrendszer sejtspecifikusan képes szabályozni egy magreceptor működését. Mindez felhívja a figyelmet a sejtek gyulladásos állapota és a lipidanyagcsere kapcsolatára olyan folyamatokban, mint pl. az érelmeszesedés. | Peroxisome Proliferator-activated Receptor y(PPARy) is a lipid-activated transcription factor that regulates lipid metabolism and inflammation, key processes in atherosclerosis and diabetes. PPARy agonists are known to regulate inflammation on multiple levels. The goal of this grant was to study how the inflammatory milieu regulates the activity of PPARy, analyze the crosstalk between inflammatory reactions and PPARy and to find the molecular mechanism how mediators of the immune system influence the receptor activity. We demonstrated that inflammatory molecules interfere with PPARg signaling. Proinflammatory molecules inhibit, while interleukin-4 (IL-4) stimulate PPARy in macrophages and dendritic cells (DCs). Activation of IL-4 signaling augments PPARg activity through a novel interaction between PPARg and Signal Transducer and Activators of Transcription 6 on the promoter of PPAR? target genes, like FABP4. As a consequence, IL-4 strongly enhances PPARy response and acts as a licensing factor by increasing the number of genes regulated and also the magnitude of the responses. Through target gene induction PPARy can be designated a positive regulator of macrophage gene expression. These findings introduce a new mechanism how inflammatory molecules modulate the activity of a nuclear receptor via cell-type specific factors and highlight the importance of the inflammatory status of cells in lipid metabolism and atherosclerosis

Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water

Fullerenes, allotropic forms of carbon, have very interesting pharmacological effects and engineering applications. However, a very low solubility both in organic solvents and water hinders their use. Fullerene C60, the most studied among fullerenes, can be dissolved in water only in the form of nanoparticles of variable dimensions and limited stability. Here the effect on the production of C60 nanoparticles by a native and denatured hen egg white lysozyme, a highly basic protein, has been systematically studied. In order to obtain a denatured, yet soluble, lysozyme derivative, the four disulfides of the native protein were reduced and exposed cysteines were alkylated by 3-bromopropylamine, thus introducing eight additional positive charges. The C60 solubilizing properties of the modified denatured lysozyme proved to be superior to those of the native protein, allowing the preparation of biocompatible highly homogeneous and stable C60 nanoparticles using lower amounts of protein, as demonstrated by dynamic light scattering, transmission electron microscopy and atomic force microscopy studies. This lysozyme derivative could represent an effective tool for the solubilization of other carbon allotropes

Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells.

Thymic epithelial cells play a crucial role in the selection of developing thymocytes. Thymocyte-epithelial cell interactions involve a number of adhesion molecules, including members of the integrin and immunoglobulin superfamilies. We found that human thymic epithelial cells synthesize an endogenous lectin, galectin-1, which binds to oligosaccharide ligands on the surface of thymocytes and T lymphoblastoid cells. Binding of T lymphoblastoid cells to thymic epithelial cells was inhibited by antibody to galectin-1 on the epithelial cells, and by two antibodies, T305 and 2B11, that recognize carbohydrate epitopes on the T cell surface glycoproteins CD43 and CD45, respectively. T lymphoblastoid cells and thymocytes bound recombinant galectin-1, as demonstrated by flow cytometric analysis, and lectin binding was completely inhibited in the presence of lactose. The degree of galectin-1 binding to thymocytes correlated with the maturation stage of the cells, as immature thymocytes bound more galectin-1 than did mature thymocytes. Preferential binding of galectin-1 to immature thymocytes may result from regulated expression of preferred oligosaccharide ligands on those cells, since we found that the epitope recognized by the T305 antibody, the core 2 O-glycan structure on CD43, was expressed on cortical, but not medullary cells. The level of expression of the UDP-GlcNAc:Gal beta 1,3GalNAc-R beta 1, 6GlcNAc transferase (core 2 beta 1, 6 GlcNAc transferase, or C2GnT), which creates the core 2 O-glycan structure, correlated with the glycosylation change between cortical and medullary cells. Expression of mRNA encoding the C2GnT was high in subcapsular and cortical thymocytes and low in medullary thymocytes, as demonstrated by in situ hybridization. These results suggest that galectin-1 participates in thymocyte-thymic epithelial cell interactions, and that this interaction may be regulated by expression of relevant oligosaccharide ligands on the thymocyte cell surface

Distinct subpopulations of enteric neuronal progenitors defined by time of development, sympathoadrenal lineage markers and Mash-1-dependence

Enteric and sympathetic neurons have previously been proposed to be lineally related. We present independent lines of evidence that suggest that enteric neurons arise from at least two lineages, only one of which expresses markers in common with sympathoadrenal cells. In the rat, sympathoadrenal markers are expressed, in the same order as in sympathetic neurons, by a subset of enteric neuronal precursors, which also transiently express tyrosine hydroxylase. If this precursor pool is eliminated in vitro by complement-mediated lysis, enteric neurons continue to develop; however, none of these are serotonergic. In the mouse, the Mash-1−/− mutation, which eliminates sympathetic neurons, also prevents the development of enteric serotonergic neurons. Other enteric neuronal populations, however, including those that contain calcitonin gene related peptide are present. Enteric tyrosine hydroxylase-containing cells co-express Mash-1 and are eliminated by the Mash-1−/− mutation, consistent with the idea that in the mouse, as in the rat, these precursors generate serotonergic neurons. Serotonergic neurons are generated early in development, while calcitonin gene related peptide-containing enteric neurons are generated much later. These data suggest that enteric neurons are derived from at least two progenitor lineages. One transiently expresses sympathoadrenal markers, is Mash-1-dependent, and generates early-born enteric neurons, some of which are serotonergic. The other is Mash-1-independent, does not express sympathoadrenal markers, and generates late-born enteric neurons, some of which contain calcitonin gene related peptide