Nicotinic acetylcholine receptors alpha4beta2 and alpha7 regulate myelo- and erythropoiesis within the bone marrow

Nicotine consumed upon smoking affects numerous physiological processes through nicotinic acetylcholine receptors, which mediate cholinergic regulation by the neuronal and endogenous acetylcholine. Consequently, nicotinic receptors are expressed in many non-excitable tissues including the blood. In spite of the documented effect of nicotine on hematopoiesis, little is known about the expression and role of nicotinic receptors in the course of blood cell differentiation. The aim of the present study was to investigate whether and how nicotinic receptors are involved in the development of myeloid and erythroid cells within the bone marrow. The presence of nicotinic receptors containing alpha4(beta2) and alpha7 subunits in the bone marrow cells of C57Bl/6 mice was shown by the binding of [125I]-alpha-bungarotoxin or [3H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluorescein-labeled alpha-cobratoxin. Both TER119+ (erythroid) and CD16+CD43med (myeloid) progenitor cells bound more alpha4-specific antibodies than their mature forms, while the binding of alpha-cobratoxin and alpha7-specific antibodies was also high in mature cells. According to morphological analysis, either the absence of alpha7-containing nicotinic receptors in knockout mice or their desensitization in mice chronically treated with nicotine decreased the number of myeloid and erythroid progenitors and junior cells. In contrast, the absence of beta2-containing receptors favored myelocyte generation and erythroid cell maturation. It is concluded that the development of both myeloid and erythroid cell lineages is regulated by endogenous cholinergic ligands and can be affected by nicotine through alpha7- and alpha4beta2-containing nicotinic receptors, which play different roles in the course of the cell maturation.     

NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase/protease I

Escherichia coli thioesterase/protease I (TEP-I) is a lipolytic enzyme of the serine protease superfamily with Ser(10), Asp(154) and His(157) as the catalytic triad residues. Based on comparison of the low-field (1)H nuclear magnetic resonance spectra of two mutants (S10G and S12G) and two transition state analogue complexes we have assigned the exchangeable proton resonances at 16.3 ppm, 14.3 ppm, and 12.8 ppm at pH 3.5 to His(157)-N(delta1)H, Ser(10)-O(gamma)H and His(157)-N(epsilon2)H, respectively. Thus, the presence of a strong Asp(154)-His(157) hydrogen bond in free TEP-I was observed. However, Ser(10)-O(gamma)H was shown to form a H-bond with a residue other than His(157)-N(epsilon2).     

Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.     

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases (MAPKs) are a family of proteins that constitute signaling pathways involved in processes that control gene expression, cell division, cell survival, apoptosis, metabolism, differentiation and motility. The MAPK pathways can be divided into conventional and atypical MAPK pathways. The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinase, and MAPK. Atypical MAPK pathways are not organized into this three-tiered cascade. MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases. The latter are referred to as MAPK-activated protein kinases. This review focuses on one such MAPK-activated protein kinase, MAPK-activated protein kinase 5 (MK5) or p38-regulated/activated protein kinase (PRAK). This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways. Recent findings on the regulation of the activity and subcellular localization, bona fide interaction partners and physiological roles of MK5/PRAK are discussed.     

Hyperspectral darkfield microscopy of PEGylated gold nanoparticles targeting CD44‐expressing cancer cells

We present a new hyperspectral darkfield imaging system with a scanned broadband supercontinuum light source. We observed the specific attachment of the functionalized gold plasmonic nanoparticles (AuNPs) targeting CD44⁺ human breast cancer cells by conventional and by proposed hyperspectral darkfield microscopy. This wide‐field and low phototoxic hyperspectral imaging system has been successful for performing spectral three‐dimensional (3D) localization and spectroscopic identification of CD44‐targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic‐based imaging, disease detection and treatment in complex biological environment. Presented system capability for 3D NP tracking will also enable investigation of specific sub‐cellular activity with the use of NPs as spectral sensors. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Angiotensin II Inhibits Satellite Cell Proliferation and Prevents Skeletal Muscle Regeneration

Cachexia is a serious complication of many chronic diseases, such as congestive heart failure (CHF) and chronic kidney disease (CKD). Although patients with advanced CHF or CKD often have increased angiotensin II (Ang II) levels and cachexia and Ang II causes skeletal muscle wasting in rodents, the potential effects of Ang II on muscle regeneration are unknown. Muscle regeneration is highly dependent on the ability of a pool of muscle stem cells (satellite cells) to proliferate and to repair damaged myofibers or form new myofibers. Here we show that Ang II reduced skeletal muscle regeneration via inhibition of satellite cell (SC) proliferation. Ang II reduced the number of regenerating myofibers and decreased expression of SC proliferation/differentiation markers (MyoD, myogenin, and active-Notch) after cardiotoxin-induced muscle injury in vivo and in SCs cultured in vitro. Ang II depleted the basal pool of SCs, as detected in Myf5^nLacZ/+ mice and by FACS sorting, and this effect was inhibited by Ang II AT1 receptor (AT1R) blockade and in AT1aR-null mice. AT1R was highly expressed in SCs, and Notch activation abrogated the AT1R-mediated antiproliferative effect of Ang II in cultured SCs. In mice that developed CHF postmyocardial infarction, there was skeletal muscle wasting and reduced SC numbers that were inhibited by AT1R blockade. Ang II inhibition of skeletal muscle regeneration via AT1 receptor-dependent suppression of SC Notch and MyoD signaling and proliferation is likely to play an important role in mechanisms leading to cachexia in chronic disease states such as CHF and CKD.     

Characterization of the neuron-specific L1-CAM cytoplasmic tail: naturally disordered in solution it exercises different binding modes for different adaptor proteins

L1, a highly conserved transmembrane glycoprotein member of the immunoglobulin superfamily of cell adhesion molecules, mediates many developmental processes in the nervous system. Here we present the biophysical characterization and the binding properties of the least structurally defined part of this receptor: its cytoplasmic tail (CT). We have shown by analytical ultracentrifugation and dynamic light scattering experiments that it is mostly monomeric and unstructured in aqueous solution. We have defined by nuclear magnetic resonance the molecular details of L1-CT binding to two major targets: a membrane-cytoskeletal linker (MCL), ezrin, and an endocytosis mediator, AP2. Surprisingly, in addition to the two previously identified ezrin binding motifs, the juxtamembrane and the (1176)YRSLE regions, we have discovered a third one, a part of which has been previously associated with binding to another MCL, ankyrin. For the L1 interaction with AP2 we have determined the precise interaction region surrounding the (1176)YRSLE binding site and that this overlaps with the second ezrin binding site. In addition, we have shown that the juxtamembrane region of L1-CT has some binding affinity to AP2-mu2, although the specificity of this interaction needs further investigation. These data indicate that L1-CT belongs to the class of intrinsically disordered proteins. Endogenous flexibility of L1-CT might play an important role in dynamic regulation of intracellular signaling: the ability of cytoplasmic tails to accommodate different targets has the potential to fine-tune signal transduction via cell surface receptors.