Cilia‐localized LKB1 regulates chemokine signaling,macrophage recruitment,and tissue homeostasis in the kidney

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy‐related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell‐autonomous manner and results in peritubular accumulation of CCR2⁺ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.     

Automated Electron Microscope Tomography of Frozen–Hydrated Chromatin: The Irregular Three-Dimensional Zigzag Architecture Persists in Compact, Isolated Fibers

The potential of electron microscope tomography as a tool for obtaining three-dimensional (3D) information about large macromolecular assemblies is greatly extended by automation of data collection. With the implementation of automated control of tilting, focusing, and digital image recording described here, tilt series of frozen–hydrated specimens can be collected with the requisite low dose. Long chromatin fibers were prepared in 90 mM monovalent ions to maintain a fully compact conformation, and after vitrification were completely contained within the ice layer. Tilt series of this material were recorded at 5° tilt increments between +60° and −60°, with a cumulative dose of ≈35 e⁻/Ųfor the series. This extremely low dose data was successfully aligned, then reconstructed by weighted backprojection. The underlying architecture of the fibers is an irregular 3D zigzag of interconnected nucleosomes, with the linker DNA between successive nucleosomes in a largely extended conformation. The visualization of this structural motif within long, frozen–hydrated chromatin fibers at relatively high salt extends our previous studies on small fragments at low ionic strength and is in agreement with the observation of this architecture in chromatin fibersin situin sectioned nuclei.     

Differential expression of the human somatostatin receptor subtypes sst1 to sst5 in various adrenal tumors and normal adrenal gland.

Somatostatin (SRIF) is a widely distributed peptide with growth-inhibiting effects in various tumors. So far, five distinct human SRIF receptor subtypes (sst1-sst5) have been identified. We investigated expression of the five ssts in various adrenal tumors and in normal adrenal gland. Tissue was obtained from ten pheochromocytomas (PHEOs), nine cortisol-secreting adenomas (CPAs), eleven aldosterone secreting adenomas (APAs) and eight non-functional adenomas (NFAs) after retroperitoneoscopic surgery, and used for RNA extraction. Adrenal tissue surrounding the tumor was available for analysis in twenty-seven cases. Receptor expression was studied by RT-PCR using sst-specific primers and subsequently confirmed by Southern blotting. Expression of all five receptor subtypes was observed in RNA obtained from normal adrenal gland. Furthermore, each receptor subtype was expressed in more than 50 % of all tumors analyzed. No sst5 expression was found in PHEOs, while sst1 was present in nearly all of these tumors. Only a few of the CPAs expressed subtypes sst1 and sst4. Expression of all five subtypes was distributed equally in APAs. No sst4 was found in any of the NFAs. Differential expression of ssts in various adrenal tumors may point to new aspects in the pathogenesis of these adenomas. Furthermore, the presence of specific ssts could expand the diagnostic and therapeutic strategies during management. New subtype specific analogues of SRIF may be used in the future depending on the type of adrenal tumor and receptor subtype expressed.     

Purification and properties of chicken prothrombin

Prothrombin was isolated from citrated chicken plasma. The isolation depends upon the elimination of an interfering substance closely adherent to chicken prothrombin by treatment with SrCO₃. Subsequent to this, the classical adsorption to barium citrate, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-200 was carried out. Prothrombin purified by this method was found to have a specific activity of 1050 Iowa units (850 N.I.H. thrombin units) per mg. Recovery from plasma averaged 40%. Molecular weight by Sephadex G-200 chromatography was 73,000 ± 5,000 and by dodecyl sulfate sodium salt acrylamide gel electrophoresis 70,000 ± 5,000. A stable dimer of M_r 138,000 was observed in some preparations. The isoelectric pH in both acetate and phosphate buffers (μ = 0.1) was 3.95. Rabbit antibody to chicken prothrombin evidenced a single line by immunoelectrophoresis against purified antigen and chicken plasma.     

Nonlinear Electrical Effects in Lipid Bilayer Membranes: III. The Dissociation Field Effect

In the course of an analysis of nonlinear electrical effects in lipid bilayer membranes, the influence of the dissociation field (or Wien) effect on the membrane conductivity is investigated. It is shown that the theory of Onsager for the Wien effect in a macroscopic phase can be applied to a thin membrane when the proper boundary conditions at the membrane-solution interface are introduced. It is assumed that an activation energy is associated with the passage of the ion across the interface. The mathematical treatment of the model is restricted to the case for which cations and anions have identical properties except for the charge sign. The resulting differential equations for the ion concentration within the membrane are integrated numerically. The analysis shows that the influence of the Wien effect on the membrane conductivity is appreciable only if the energy barrier at the interface is sufficiently high, i.e. if the rate limiting step for the ion transport is the passage of the ion across the interface.     

Structural organization of a full-length gp130/LIF-R cytokine receptor transmembrane complex

gp130 is a shared receptor for at least nine cytokines and can signal either as a homodimer or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here, we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-Ralpha). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6Ralpha hexameric complex, CNTF/CNTF-Ralpha heterodimerizes gp130 and LIF-R via noncooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic analysis of the full-length gp130/LIF-R/CNTF-Ralpha/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the "tall" class of gp130 family cytokine receptor complexes including LIF, IL-27, IL-12, and others.     

Morphological and functional characterization of the thoracic portion of blowfly salivary glands

The abdominal portion of the salivary glands in the blowfly has been studied intensively. Here, we examine the thoracic part of the salivary glands, emphasizing structural and functional aspects. The initial segment downstream of the abdominal portion is secretory and resembles the latter in most structural and functional aspects: the apical membrane is enfolded, forms a canalicular system and contains V-H⁺-ATPase that assembles upon stimulation with the hormone serotonin (5-HT); Na,K-ATPase is localized in the basolateral membrane; septate junctions are not prominent, as deduced from immunofluorescence staining for the marker proteins discs large and fasciclin III. 5-HT elicits, at low concentrations, cytoplasmic [Ca²⁺] oscillations, and, at saturating concentrations, a tonic [Ca²⁺] rise. The following, so-called “re-absorptive” segment loops through the coiled secretory portion of the salivary gland. The apical membrane of the re-absorptive cells is not enfolded, and septate junctions are prominent. V-H⁺-ATPase and Na,K-ATPase reside on the apical and basolateral membranes, respectively. Finally, re-absorptive cells are also sensitive to 5-HT; however, whereas V-ATPase assembly has a 5-HT concentration dependence similar to other segments, the Ca²⁺ response occurs only at higher 5-HT concentrations, and displays a different kinetic pattern.     

Synthesis of Dideoxymycobactin Antigens Presented by CD1a Reveals T Cell Fine Specificity for Natural Lipopeptide Structures

Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected α-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.Pathogens are detected by the host when antigenic molecules directly contact immune receptors during the early stages of infection. The strategy of intracellular infection allows viruses, certain bacteria and protozoa to partially cloak themselves from the immune response by physically encapsulating their antigens within host cells. Intracellular residence also takes advantage of immune tolerance mechanisms that prevent autoimmune destruction of self. T cells play a central role in immunity to intracellular pathogens because they can respond to antigens that are generated inside cells and then transported to the surface of infected cells after binding to antigen-presenting molecules. The antigen-presenting molecules encoded in the major histocompatibility complex are widely known for presenting peptide fragments of proteins (1). More recently, human and mouse members of the CD1 (cluster of differentiation 1) system have been shown to present small amphipathic molecules, including a variety of membrane lipids, glycolipids, and lipopeptides, greatly expanding the molecular structures recognized by the cellular immune system (2, 3).Among human CD1 proteins (CD1a, CD1b, CD1c, CD1d, and CD1e), each CD1 isoform is expressed on a different spectrum of antigen-presenting cells. Human CD1a proteins are distinguished from other CD1 proteins by high expression levels on the surface of intradermal Langerhans cells, which play a role in barrier immune function (4). Human T cell clones have been shown to directly recognize CD1a proteins in the presence of exogenous foreign antigens (5) or in the presence of sulfatide and other self lipids (6, 7), suggesting a role for CD1a in T cell activation. In addition, mycobacteria and other intracellular pathogens have been shown to increase CD1a expression in lesions found in leprosy and tuberculosis patients, implying a possible role for CD1a in the response to infection, especially at mucosal or skin sites (8–10). Analysis of the molecular target recognized by CD1a-restricted T cell clone (CD8-2) allowed the identification of a foreign antigen presented by CD1a as dideoxymycobactin (DDM) (11).²Mycobactin binds iron to promote Mycobacterium tuberculosis survival. DDM was initially isolated (11) from antigenic lipid extracts of M. tuberculosis, a pathogen that kills ∼1.7 million humans annually on a worldwide basis (12). The determination of DDM structure was based on mass spectrometric and NMR studies of limiting amounts of natural material derived from the pathogenic organisms, so that not all elements of its chemical structure could be formally determined. Instead, its assigned structure was facilitated by obvious parallels of dideoxymycobactin with mycobactin, a lipopeptide siderophore (13, 14). Iron is required for reduction-oxidation reactions involving respiration and other basic metabolic pathways in bacterial pathogens (13). Environmental mycobacteria have at least two iron uptake pathways, but mycobactin and the related molecule carboxymycobactin represent the only known dedicated iron uptake pathway for pathogenic species like M. tuberculosis (15, 16). Highlighting the physiological importance of the mycobactin pathway, deletion of mycobactin synthase B limits M. tuberculosis survival in cells (13, 14). Also, mammalian innate immune systems produce siderocalin, a 20-kDa lipocalin that binds both ferric and apo siderophores, preventing their uptake and subsequent iron delivery to microbes (17–20). The small available yields of natural material highlighted the need for a straightforward method to synthesize DDM for studies of its role in mycobacterial iron acquisition and testing T cell responses in human populations, as well as to provide authentic standards to investigate unknown aspects of natural DDM stereochemistry. Here we report two syntheses for production of DDM in solution phase and solid phase. Comparison of synthetic and natural DDMs gives unexpected insight into the stereochemical structures of the methylserine, oxazoline, and butyrate moieties of DDM and provides direct evidence that the T cell response is highly specific for a unique aspect of DDM structure that protrudes from the surface of the CD1a-DDM complexes.