Phillip E. Ryals

Evidence for Early Signaling Events in Stomatin‐Induced Differentiation of Tetrahymena vorax

ABSTRACT The mechanism of stomatin‐induced differentiation of Tetrahymena vorax was investigated by in vivo protease degradation of cell surface proteins, the direct measurement of products formed from the activation of phospholipase C, and the use of an array of signal transduction inhibitors/activators. The data indicate that a surface‐exposed protein is required for stomatin to signal the cells to differentiate and that the cells are committed to the differentiation pathway within two hours after exposure to stomatin. Analysis of radiolabeled polyphosphoinositols and inositol lipids from control and stomatin‐treated populations in the presence of 10 mM LiCl were consistent with a rapid activation of phospholipase C. Within five min following addition of stomatin, this resulted in an increase in polyphosphoinositols and a concomitant decrease in the relative amounts of phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate.

Phenotype Switching in Polymorphic Tetrahymena: A Single-Cell Jekyll and Hyde

For nearly half a century, phenotype switching in the group of polymorphic species of the ciliate genus Tetrahymena has been the subject of investigations of the underlying mechanisms, the accompanying biochemical and structural changes, and the evolution of polymorphic survival strategy. Beginning with the pioneering systematic studies by Furgason in 1940 of hymenostome ciliates, the experimental approach rapidly expanded to include investigations of growth, nutrition, physiology, morphology, and morphogenesis in the polymorphic species. Recently, with progress in elucidation of the novel signaling ligand and identification of elements of the subsequent signal transduction cascade, in addition to the growing catalog of intracellular events associated with differentiation in these unicellular eukaryotes, we have begun to address the mechanistic basis of polymorphism. This review summarizes and integrates the history and recent discoveries concerning Tetrahymena polymorphic cells. We are now poised to answer fundamental questions about this interesting pathway of cell differentiation.

Derivation of extracellular polysaccharide-deficient variants from a serotype A strain of Pasteurella multocida

Abstract. The production of serotype A extracellular polysaccharide is thought to be associated with expression of an approximately 40-kDa lipoprotein (Plp-40) present on the outer surface of Pasteurella multocida strains of avian origin. The tendency of certain strains to undergo colonial dissociation concomitantly with serial passaging on laboratory growth media was exploited to derive two variant strains exhibiting the capsule-deficient phenotype from a heavily capsulated parental strain. Assessments of colonial consistency, iridescence, gentian violet binding, and hyaluronidase sensitivity were consistent with cellular observations indicating little or no capsulation of derivative strains. Fluorographic analysis of electrophoretically resolved cellular lipoproteins labeled with [3H]-palmitate revealed capsular loss occurred with a concomitant diminution of Plp-40 production in the variant strains. In contrast, a phenotypically stable strain that did not undergo colonial dissociation under identical conditions exhibited no decrease in Plp-40 content. This work provides a model system for investigating the role of extracellular polysaccharide in the cell surface physiology and pathogenicity of P. multocida. The present results strongly support the notion that Plp-40 is associated with serotype A capsular material and suggest coordinate regulation of their biosynthesis.

Relationship Between Serotype A Encapsulation and a 40-kDa Lipoprotein in Pasteurella multocida

Abstract. Eleven serotype A encapsulated and nonencapsulated strains of Pasteurella multocida were examined with regard to lipoprotein content. Relative amounts of an approximately 40-kDa lipoprotein (Plp-40) were found to correlate directly with the degree of encapsulation in that heavily encapsulated strains exhibited the greatest amounts, while nonencapsulated strains possessed little or no Plp-40.

Prevalence of a novel capsule-associated lipoprotein among Pasteurellaceae pathogenic in animals

Capsular serotype A strains of Pasteurella multocida of avian origin express a 40-kDa lipoprotein (Plp-40) thought to attach the extracellular polysaccharide to the cell surface. The objective of the present study was to assess the prevalence of Plp-40 in P. multocida strains of disparate serotypes and host origins, as well as other pathogenic members of the family Pasteurellaceae. Exponential-phase reference and clinical isolates were radiolabeled with [3H]-palmitate, lysed to obtain whole-cell protein fractions, and analyzed using SDS-PAGE and fluorography to assess lipoprotein content. The ability to produce Plp-40 was found to be conserved among certain P. multocida reference and clinical strains of different host origins including avian, human, porcine, bovine, feline, canine, ovine, and cervine, but not rabbit. Production of a 40-kDa lipoprotein was exhibited by all clinical isolates of Pasteurella aerogenes, Pasteurella pneumotropica, Actinobacillus suis, Actinobacillus suis-like organism, and Actinobacillus pleuropneumoniae examined, but not Pasteurella (Mannheimia) haemolytica, Actinobacillus lignieresii, or Haemophilus spp. These data suggest that, while not all Pasteurellaceae are able to produce a 40-kDa lipoprotein under the present experimental conditions, expression is somewhat conserved among diverse isolates of disparate host origins.

Effects of Dichloroisoproterenol on Macromolecular Synthesis and Differentiation in Tetrahymena vorax

ABSTRACT. The effect of dichloroisoproterenol on macrostomal cell formation in Tetrahymena vorax was examined and the drug was found to be 50% inhibitory at a concentration of 88 μM. Cellular uptake and incorporation of a variety of radiolabelled precursors was monitored in the presence of dichloroisoproterenol. The results demonstrate a strong, concentration‐dependent inhibitory effect on RNA and protein biosynthesis, with a lesser inhibition observed for lipid biosynthesis. These data indicate that dichloroisoproterenols reported effects on vacuole formation and processing in Tetrahymena are nonspecific with regard to phagocytic processes, but result from a general suppression of macromolecular synthesis.

Protein Prenylation in Tetrahymena

Summary Tetrahymena vorax V 2S and Tetrahymena pyriformis GL were examined for the presence of proteins covalently modified with mevalonate-derived lipids. Radiolabeling of cells using [3H]mevalonolactone, [ 3 H]mevalonate, [ 3 H]farnesyl pyrophosphate or [ 3 H]geranylgeranyl pyrophosphate resulted in the appearance of protein-associated radioactivity following protein delipidation in acetone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled protein revealed radioactive polypeptides ranging in molecular mass from 123 to 14 kDa following scintillation counting of gel slices or fluorography. Methyl iodide cleavage of total labeled protein followed by HPLC analysis and scintillation counting of the released prenyl alcohols showed radioactive peaks that co-chromatographed with commercially obtained farnesol (C 15 ) and geranylgeraniol (C 20 ).

Tetrahymena thermophila disA mutants exhibit aberrant levels of phosphatidylethanolamine

Summary Tetrahymena thermophila wild type strain B 1868 and T. thermophila disA mutant were examined for total phospholipid composition by radiolabeling whole cells with [ 32 P]orthophosphate, [ 3 H]myristic acid, and [ 3 H]acetate. Percent distribution of radioactivity into individual phospholipid classes was determined by scintillation counting or by autoradiography following chromatography of total lipid extracts on silica gel thin layer plates. A significant reduction in radiolabel incorporation into the disA mutant phosphatidylethanolamine fraction was observed in all radiolabeling experiments.