ABSTRACT
Although sigma 1 (σ(1)) receptors and mitogen-activated protein kinases (MAPKs) are known modulators of neuroprotection, a role for MAPK signaling pathways in σ receptor-mediated neuroprotection has not been investigated in detail.The present study aims to investigate the possible link between σ(1) receptors and MAPKs in neuroprotection. Primary mixed cortical and hippocampal neurons were treated with σ(1) receptor agonists PRE-084 or 4-PPBP in a time- and concentration-dependent manner; and in another set of experiments, cells were pre-incubated with σ(1) receptor antagonist BD1047 or MEK inhibitor PD98059 in a concentration-dependent manner prior to PRE-084 or 4-PPBP treatment. Levels of phosphorylated and total ERK1/2, JNK and p38-MAPK were determined with western blotting and ERK1/2 phosphorylation was confirmed with immunofluorescence. To investigate neuroprotection by σ(1) receptors, cells were pre-treated with PRE-084 or 4-PPBP and glucose-starved for various times: in the presence or absence of pre-incubated BD1047 or PD98059. Cell viability was then measured with MTT assay. Both PRE-084 and 4-PPBP caused phosphorylation of ERK1/2, but not p38-MAPK and JNK. ERK1/2 phosphorylation was inhibited by BD1047 and PD98059 in a concentration-dependent manner. Immunofluorescence confirmed the phosphorylation of ERK1/2 by PRE-084 and 4-PPBP and its inhibition by BD1047 and PD98059. Pre-treating glucose-deprived neurons with 4-PPBP, but not PRE-084; caused neuroprotection which was inhibited by BD1047 and PD98059. 4-PPBP, but not PRE-084; causes ERK1/2 phosphorylation-mediated neuroprotection. This presents a novel mechanism by σ(1) receptors in modulating neuroprotection.
Subject(s)
Haloperidol/analogs & derivatives , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Neurons/drug effects , Phosphorylation/drug effects , Receptors, sigma/metabolism , Analysis of Variance , Animals , Blotting, Western , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Dose-Response Relationship, Drug , Fluorescent Antibody Technique , Haloperidol/pharmacology , Morpholines/pharmacology , Neurons/metabolism , Neuroprotective Agents/pharmacology , Receptors, sigma/agonistsABSTRACT
Interleukin 18 (IL-18) is a cytokine capable of induction of IFNgamma, granulocyte monocyte-colony stimulating factor (GM-CSF), TNFalpha and IL-1 in immunocompetent cells. Equine and feline plasmid vectors expressing pro-IL-18, mature IL-18 and IL-18 fused to a synthetic signal sequence from human IL-1beta receptor antagonist protein (ILRAP), ILRAP-IL-18, have been generated. In vitro protein expression of these constructs was compared by Western blot analysis. These data demonstrated that ILRAP-IL-18 protein was secreted readily from transfected chinese hamster ovary (CHO) cells. A simple bioassay for human IL-18 was recently described using human myelomonocytic KG-1 cells, which produce human IFNgamma in response to human IL-18 in a dose dependent manner (Konishi et al., 1997). We demonstrated bioactivity of equine and feline IL-18 protein in transfection products of CHO cells using this assay. Bioactivity of ILRAP-IL-18 protein was demonstrated in the culture medium of transfected CHO cells. These data imply that the ILRAP-IL-18 construct shows potential for use in vivo, where cell secretion of protein is crucial.
Subject(s)
Cats/immunology , Gene Expression Regulation/immunology , Horses/immunology , Interleukin-18/biosynthesis , Animals , Biological Assay , CHO Cells , Cricetinae , Cricetulus , In Vitro Techniques , Interferon-gamma/biosynthesis , Interleukin 1 Receptor Antagonist Protein , Interleukin-18/analysis , Recombinant Fusion Proteins/biosynthesis , Sialoglycoproteins/geneticsABSTRACT
Streptococcus gallolyticus (S. caprinus) was resistant in vitro to at least 7% (w/v) tannic acid and 4% (w/v) acacia condensed tannin, levels 10-fold greater than those tolerated by S. bovis. Growth of S. gallolyticus in liquid medium was characterized by a lag period which increased, and a growth rate which decreased, with increasing tannin concentration. S. gallolyticus was also more tolerant to the presence of simple phenolic acid monomers than was S. bovis, but the lag period was still concentration dependent. Gallate decarboxylase activity in S. gallolyticus was elevated in the presence of tannic acid or gallic acid but not with other phenolic acids. Scanning electron microscopic analysis showed that both the size and shape of S. gallolyticus and S. bovis changed in response to tannin but only S. gallolyticus was surrounded by an extracellular polysaccharide matrix which accumulated in a tannin-concentration-dependent fashion. Washing of the cells to remove extracellular polysaccharide increased the lag period of S. gallolyticus in the presence of 1% (w/v) tannic acid from 4 h to 6 h. In contrast, increasing extracellular polysaccharide synthesis in S. bovis did not increase its tolerance to tannic acid. These data demonstrate that S. gallolyticus has developed a number of mechanisms to reduce the potential effect of tannins on cell growth, and that these mechanisms provide the organism with a selective advantage over S. bovis when grown in the presence of tannins.
Subject(s)
Hydrolyzable Tannins/metabolism , Streptococcus bovis/metabolism , Streptococcus/metabolism , Carboxy-Lyases/metabolism , Cell Division , Culture Media , Extracellular Matrix/metabolism , Gallic Acid/metabolism , Hydrolysis , Microscopy, Electron, Scanning , Polysaccharides, Bacterial/biosynthesis , Streptococcus/growth & development , Streptococcus/ultrastructure , Streptococcus bovis/growth & development , Streptococcus bovis/ultrastructureABSTRACT
The damaging effects of UVB light have been described previously and include a number of changes to multiple cell types. At previous meetings of this society, we have shown that Langerhans' cells are the most susceptible to UVB induced damage which can be shown as ultrastructural changes in dendrites, nucleus and cytoplasm by transmission electron microscopy. We have also shown that their patterns of migration from skin to regional lymph node and their ability to present antigens to autologous T cells have been profoundly altered by UVB irradiation. The aim of this work was to establish if it was possible to reverse any of the damage done to Langerhans' cells by UVB exposure by topical application of a DNA repair enzyme such as T4N5 endonuclease. These experiments were undertaken in a sheep model that allowed collection of cells as they migrate from the skin. This allowed for a direct examination of the migration characteristics and ultrastructural features of all Langerhans' cells before, during, and for 2 weeks after exposure to a single dose of UVB. Results obtained from this project indicate that treatment by topical application of DNA repair enzyme immediately after UVB irradiation may restore a number of normal immune parameters associated with the structure and function of migrating Langerhans' cells. It appears that there is a dose related correction of the increased tempo of cell migration and some improvements in the number of ultrastructurally damaged Langerhans' cells have also been associated with application of higher doses of DNA repair enzyme. These preliminary findings indicate that some potential therapeutic benefits are associated with the use of such agents in reversing the immunological damage caused by exposure to erythemal doses of UVB light.
Subject(s)
Skin/radiation effects , Ultraviolet Rays/adverse effects , Animals , Cell Movement/radiation effects , Langerhans Cells/pathology , Langerhans Cells/radiation effects , Langerhans Cells/ultrastructure , Lymph Nodes/physiology , Sheep , Skin/cytology , Skin/pathology , Time FactorsABSTRACT
Our past work has shown that long, flexible type IV pili (single or in bundles) are the predominant pili expressed on fecal isolates of diarrhea-associated species of Aeromonas (Aeromonas veronii biovar sobria and A. caviae). They represent a family of type IV pili which we have designated Bfp (for bundle-forming pili). Reports from Japan suggest that Bfp are intestinal colonization factors. This study presents compelling evidence to support this conclusion. Aeromonas bacteria and/or Bfp purified from a strain of A. veronii biovar sobria were shown to adhere to epithelial and intestinal cell lines, freshly isolated human enterocytes, and fresh and fixed human and rabbit intestinal tissues, as determined by light and electron microscopy and immunohistochemical detection. Removal of Bfp by mechanical means decreased adhesion to cell lines by up to 80%. Purified Bfp blocked adhesion of the test strain to intestinal cells in a dose-dependent manner. Adhesion was also blocked by the Fab fraction of anti-Bfp immunoglobulin G. Moreover, ultrastructural studies (ruthenium red staining and transmission and scanning electron microscopy) demonstrated for the first time that Aeromonas adhesion to human enterocytes is pilus mediated and suggested that Bfp may also promote colonization by forming bacterium-to-bacterium linkages. Bfp-positive isolates examined for type IV pilus-mediated twitching motility in agar and slide culture assays developed for Pseudomonas aeruginosa did not, however, exhibit this function.