Constitutive expression of IL-22 in the human intervertebral disc and its reduction by exposure to pro-inflammatory cytokines in vitro

The importance of cytokines in disc degeneration is well recognized. Little is known about IL-22 expression in the human intervertebral disc. We investigated IL-22 immuno-localization in disc tissue, and molecular expression and production of IL-22 by annulus cells cultured in three-dimensional (3D) culture. We examined human disc tissue using immunohistochemistry and we cultured isolated annulus cells in 3D to analyze IL-22 expression and production, and its receptor, IL-22R, in conditioned media. Ingenuity pathway analysis (IPA) also was used to identify significant gene expression networks within the molecular data. IL-22 and IL-22R were immunolocalized in many cells in the human outer and inner annulus; fewer cells exhibited localization in the nucleus. Three-dimensional culture of annulus cells demonstrated production of IL-22 in conditioned media; exposure to IL-1ß or TNF-α significantly reduced IL-22 levels. Significant decreases also were identified in conditioned media assayed for IL-22R in TNF-α treated cells. IPA analysis showed that IL-22 ranked among the top canonical pathways. We found constitutive expression and production of IL-22 and IL-22R in the disc, which expands our understanding of the effect of pro-inflammatory cytokines on IL-22 expression and production. Three-dimensional cultured annulus cells exposed to IL-1ß or TNF produced significantly lower levels of IL-22 into their conditioned media compared to levels produced by control cells. Our findings have clinical relevance because of the elevated pro-inflammatory milieu within the degenerating human disc.     

Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State

Fad35R from Mycobacterium tuberculosis binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.     

An early Miocene deep‐water decapod crustacean faunule from the Vienna Basin (Western Carpathians,Slovakia)

Abstract: A decapod crustacean faunule from the lower Miocene (upper Burdigalian, ‘Karpatian’) of the Slovakian part of the Vienna Basin comprise five new species: Callianopsis marianae (Ctenochelidae), Crosniera schweitzerae (Thomassiniidae), Agononida cerovensis and Munidopsis lieskovensis (both Galatheidae) plus Mursia harnicari (Calappidae). The new species of Callianopsis is the first undoubted member of the genus to be recorded from Europe; it is based on sexually dimorphic major and minor chelae as well as on portions of carapace and abdomen. Crosniera schweitzerae sp. nov. and Agononida cerovensis sp. nov. constitute the first fossil members of these genera. Additional material of an enigmatic crab, Styrioplax exiguus, and a re‐examination of the type material, confirms assignment of that genus to the subfamily Rhizopinae (family Pilumnidae). Palaeoecological data suggest that deposition of the levels (Lak?árska Nová Ves Formation) from which these taxa were collected took place under generally low‐energy, deep‐water conditions that were conducive to the preservation of delicate structures. Palaeobiogeographical affinities of the described taxa suggest a trans‐Atlantic migration during the early Miocene.     

Cost-effectiveness of a structured progressive task-oriented circuit class training programme to enhance walking competency after stroke: The protocol of the FIT-Stroke trial

Background  

Most patients who suffer a stroke experience reduced walking competency and health-related quality of life (HRQoL). A key factor in effective stroke rehabilitation is intensive, task-specific training. Recent studies suggest that intensive, patient-tailored training can be organized as a circuit with a series of task-oriented workstations.     

The use of flow microfluorimetry in the analysis of the phenotype expression of mouse histocompatibility antigens

Quantitation of the expression of cell surface antigens has hitherto been limited to analysis by either cytotoxicity tests or radioimmune assays (5, 15). We report here the use of a new methodology to analyze and quantitate the expression of mouse histocompabililty antigens (H-2 locus) in hybrid clones and parental cell types. The binding of fluorescein-tagged antibody is measured on a cell-to-cell basis in large viable cell populations using flow microfluorimetric techniques. These techniques have been used to measure hapten and immunoglobulin binding to lymphocyte populations (8, 9, 14). However, this is the first report in which these techniques have been used to examine the expression of the H-2 locus. The advantage of this approach is twofold: first, a large and statistically significant sample population may be analyzed one cell at a time, thus revealing the fine detail of heterogeneity in the expression of the cell surface markers within a population. Second, as has been demonstrated for analysis of specific components of the immune system, this method does permit fluorescence-activated sorting of cell types according to their different surface populations (8, 9, 14).     

Occurrence of secretory structures in underground systems of seven Asteraceae species

In contrast with the abundance of anatomical studies of secretory structures on aerial vegetative organs of Asteraceae species, the information about secretory structures on thickened subterranean organs is sparse. The aim of this study was to investigate the occurrence of secretory structures on thickened and nonthickened subterranean organs of seven Asteraceae species from three tribes: Eupatorieae (Chromolaena squalida and Gyptis lanigera), Vernonieae (Chresta sphaerocephala, Lessingianthus bardanoides, L. glabratus and Orthopappus angustifolius), and Plucheeae (Pterocaulon angustifolium). The specimens were collected in areas of cerrado from the State of São Paulo, Brazil. All species of the tribe Vernonieae studied exhibited endodermic cells, other than the epithelial cells of the canal, with secretory activity in the roots. In C. sphaerocephala roots, two types of endodermic cell were found, but only one had secretory activity. Secretory canals were found in the tuberous and nontuberous roots of all studied species. These data agree with the results from the literature for Asteraceae species. Here, we describe for the first time in Asteraceae the presence of secretory idioblasts in C. sphaerocephala. Secretory trichomes are present in the Orthopappus angustifolius rhizophore. Histochemical tests have shown that all types of secretory structure possess substances containing lipids. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157 , 789–796.     

Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function

The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins. When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina. Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact. The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively. Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B. LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices. Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface. Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca. 30 nm. These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.