The Effect of 2-Mercapto-1-(Beta-4-Pyridethyl) Benzimidazole (MPB) on Cell Differentiation and RNA Synthesis in the Protozoon Tetrahymena vorax1

SYNOPSIS. Differentiation of small-mouthed cells (microstomes) into large-mouthed, potentially carnivorous cells (macrostomes) in Tetrahymena vorax is prevented by 2-mercapto-1-(β-4-pyridethyl) benzimidazole (MPB). This differentiation, induced by the transforming principle, stomatin, isolated from the potential prey, Tetrahymena pyriformis, is a synchronous process in which 70–95% of the population of T. vorax microstomes transform into macrostomes within 450 min. MPB also inhibits RNA synthesis in transforming microstomes while having little effect on protein synthesis. Finally, the effect of MPB on both transformation and RNA synthesis is reversible.     

An Analysis of Macrostome Production in Tetrahymena vorax Strain V2S-Type*

SYNOPSIS. Tetrahymena vorax is a small polymorphic holotrichous ciliate capable of transforming to macrostomes when microstomes are washed and suspended with prey in distilled water. Extrinsic factors having an effect on this transformation were examined; maximum yields of macrostomes (in excess of 90%) were obtained under the following conditions: populations of both prey, T. pyriformis, and potential predator, T. vorax microstomes, were grown on Loefer's medium for 48 hours prior to washing in distilled water. The density of the prey was adjusted to 300,000 cells/ml and the predator density to 2,000–3,000 cells/ml. Five ml of prey suspension and 5 ml of T. vorax microstome suspension were mixed together in a large petri dish because a high surface-volume ratio is important for high yields of macrostomes. The pH was adjusted to 6.0 and the petri dish was placed at 20 C for 12 hr. Macrostomes then appeared about 6 hr after addition of the prey. A dialyzable, heat stable substance released by the prey which can induce the microstome-macrostome transformation was isolated. This material was effective after being stored for weeks in the cold; its activity was not affected by the protein digesting enzymes pepsin or trypsin. This factor was called stomatin because its first visible effect in producing microstome-macrostome transformation appeared to be to incite reorganization of the oral structures.     

Précisions Temporelles sur les Processus de la Stomatogenèse de Tetrahymena paravorax analysés en Microscopie Photonique

Resume. Une analyse des séquences morphogénétiques du CiliéTetrahymena paravorax montre que: (A) La durée de la stomatogenèse de bipartition des formes microstomes en croissance exponentielle représente 45% du temps de génération (stade 1—20%; stade 2—3%; stade 3—3%; stade 4—5%; stade 5—5%; stade 6—9%). (B) La division cytoplasmique est inégale (les proters sont plus petits que les opisthes); la différence de taille initiale entre les 2 produits de fission est probablement compensée par une prolongation de la période de croissance chez le proter. (C) Le pourcentage maximum de réorganisation buccale microstome → macrostome pour les populations asynchrones atteint –? 70% au bout de 210 mn d'incubation dans la stomatine. (D) L'initiation de la stomatogenèse de remplacement oral est connectée avec la fin d'une période dont la durée minimale est approximativement celle du stade 0 du cycle normal d'interdivision des microstomes; cette initiation est retardée chez les microstomes exposés à la stomatine dès le début du cycle cellulaire. (E) Le primordium buccal de division peut se résorber en présence de stomatine et la stomatogenèse antérieure peut commencer avant que ne soit terminée cette résorption; la résorption n'est plus induite au-delà d'un point du stade 5 qui précède le début de la constriction du corps cellulaire. SYNOPSIS. An analysis of the morphogenetic sequences in the ciliate Tetrahymena paravorax has shown that: (A) The duration of predivision stomatogenesis in exponentially growing microstomes occupies 45% of the generation time (stage 1—20%; stage 2—3%; stage 3—3%; stage 4—5%; stage 5—5%; stage 6—9%). (B) Cytoplasmic division is unequal (the proters are smaller than opisthes); the initial size difference between the 2 fission products is presumably compensated by an increased growth period in the proter. (C) The maximum percentage of microstome-to-macrostome oral reorganization is –? 70% in asynchronous populations, 210 min after suspension in stomatin. (D) Initiation of oral replacement stomatogenesis is associated with the end of a period which has a minimum duration nearly equal to that of stage 0 characteristic of the normal inter-division cycle of the microstomes; this initiation is delayed if exposure of microstomes to stomatin is begun at the onset of the cell cycle. (E) The buccal primordium formed in division can be resorbed in presence of stomatin and anterior stomatogenesis can start before the resorption is completed: this resorption is not induced if the cells have progressed beyond a point which precedes the beginning of the cell furrowing (stage 5).     

Preuves Cinétiques de l'Existence d'une Période de Compétence pour la Transformation Microstome-Macrostome dans le Cycle Cellulaire de Tetrahymena paravorax

RÉSUMÉ. Des expérimentations portant sur des cellules isolées ont montré que 77% des microstomes de Tetrahymena paravorax, prélevés au hasard dans des cultures en phase logarithmique de croissance, se transforment directement en macrostomes en présence de “stomatine.” Ces macrostomes apparaissent à des moments variés entre 1,5 et 9 h après l'addition de la stomatine (“point 50% de transformations” vers 2,5 h). La compétence pour le remplacement oral est en relation avec la position dans le cycle cellulaire. Les pourcentages de transformation les plus élevés sont observés avec les populations testées pendant la première moitié de la période moyenne d'interfission. La formation des macrostomes est d'autant plus rapide que l'ǎge initial des microstomes est plus proche du point médian du cycle cellulaire (“point de compétence”). Dans la seconde moitié de ce cycle, le temps moyen de transformation reste à peu près constant, mais le pourcentage de divisions augmente: le “point de transition” (50% de divisions) se trouve au début d'une phase terminale représentant 19–20% de la durée totale du cycle. La transformation des produits de bipartition antérieur et postérieur est nettement asynchrone: dans la majorité des paires cellulaires, l'opisthe se transforme avant le proter. Les cellules-soeurs se divisent aussi de manière asynchrone: le temps de génération du proter est plus long que celui de l'opisthe. Le problème de l'acquisition de la compétence pour le changement de phénotype est discuté en envisageant les corrélations éventuelles avec certains processus majeurs du cycle cellulaire. SYNOPSIS. Seventy-seven percent of the microstomes of Tetrahymena paravorax taken from random samples of log-phase cultures transform directly into macrostomes in the presence of “stomatin.” These macrostomes appear between 1.5 and 9 h after addition of stomatin (“50% transformation point,”～ 2.5 h). Competence for oral replacement is related to the position in the cell cycle. The highest percentages of transformation are observed in populations tested during the first half of the mean interfission period. Formation of macrostomes is more rapid when the initial age of the microstomes is nearer to the midpoint of the cell cycle (“competence point”). In the 2nd half of this cycle, the mean transformation time remains approximately constant, but the percentage of cells undergoing division is increasing. The “transition point” (50% divided cells) is found at the beginning of a terminal phase which accounts for 19–20% of the cell cycle. Transformation of anterior and posterior fission products is fairly asynchronous; in the majority of individual pairs, the opisthe is transformed before the proter. The daughter cells also divide asynchronously, the generation time of the proter being longer than that of the opisthe. The problem of acquisition of competence for phenotypic change is discussed in light of possible correlations with certain major processes of the cell cycle.     

The Effects of Purine and Pyrimidines Upon Transformation in Tetrahymena vorax,Strain V2S1

Column chromatography with Biogel P2 (molecular exclusion of 1800 daltons) indicates that the transforming principle causing microstomes to become macrostomes is a small molecule. Absorbance tests show that only those fractions with high absorbance at 260 nm have biological activity, indicating that the active principle is a component of nucleic acids. Tests of purines and pyrimidines show that purines are active, with hypoxanthine having the highest activity. The combination of hypoxanthine with uridine shows a synergistic reaction. As these two compounds are the natural catabolic excretory products from nucleic acids in Tetrahymena, the fact that they induce transformation in concentrated, starving cells may be a survival mechanism allowing cannibalism to be induced when nutrients are depleted, thereby allowing the survival of the transformed cells until such time as adequate nutritional conditions are restored.     

The Formation and Fate of Tetrahymena patula Cryptostomes

A reliable method for producing reproductive cysts in Tetrahymena patula is described. The procedure involves the isolation of macrostomes without cytopharyngeal pouches in microdrops of distilled water under oil. The study of silver-impregnated specimens has shown that a complex pattern of oral resorption and reformation occurs within the cyst that leads to the formation of a group of small cells with recessed oral apparatuses. These cells, called “cryptostomes,” swim very rapidly on excystment and are incapable of either feeding or reproducing. They are presumably dispersal forms. Oral morphogenesis during the transformation of excysted cryptostomes into microstomes and macrostomes is also described.     

Life Cycle, Morphology, Ontogenesis, and Phylogeny of Bromeliothrix metopoides nov. gen., nov. spec., a Peculiar Ciliate (Protista, Colpodea) from Tank Bromeliads (Bromeliaceae)

Bromeliothrix metopoides was discovered in tank bromeliads from Central and South America. Pure cultures could be established in various media stimulating growth of its food, i.e. bacteria and heterotrophic flagellates of the genus Polytomella. The new ciliate was investigated in the light- and scanning electron microscope, with various silver impregnation techniques, and with molecular methods, using the small-subunit rDNA. The morphology and its changes during the life cycle are documented by 167 figures and a detailed morphometry. Bromeliothrix metopoides is about 27-55 × 22-36 μm in size and has a complex life cycle with Metopus-shaped, bacteriophagous theronts and trophonts (microstomes) and obovate, flagellate-feeding macrostomes having a large, triangular oral apparatus. The thin-walled resting cysts of the theronts and trophonts are uniquely ellipsoidal, while the thick-walled cyst of the macrostome morph is globular. Reproduction occurs in freely motile condition either by binary fission or polytomy, producing a unique, motile "division chain" composed of four globular offspring, of which the central ones are connected by a curious, plug-like holdfast. Division is associated with a complete reorganization of the parental oral and somatic infraciliature. Stomatogenesis is merotelokinetal as in other members of the order Colpodida. The right polykinetid is generated by the rightmost postoral kinety, while the left polykinetid is produced by the two left postoral kineties and five left side kineties. The division in freely motile condition resembles the Exocolpodidae Foissner et al., 2002, to which Bromeliothrix is tentatively assigned, differing from Exocolpoda mainly by the formation of a macrostome morph and a division chain. Bromeliothrix has a ciliary and silverline pattern typical for members of the family Colpodidae. This matches the molecular classification which, however, hardly reflects the outstanding division and life cycle, suggesting some decoupling of morphological and molecular evolution. The specific morphological and ontogenetic traits of Bromeliothrix are interpreted as adaptations to the highly competitive habitat, favouring r-selected life strategies. Bromeliothrix metopoides is widespread in various tank bromeliads and can be easily cultivated in a wide variety of limnetic and terrestrial media. Thus, it remains obscure why this ciliate is restricted to tank bromeliads, i.e. did not occur in about 2,000 soil and freshwater samples investigated globally, including some 100 samples from Central and South America.     

Electrophysiological Properties of the Microstome and Macrostome Morph of the Polymorphic Ciliate Tetrahymena vorax

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately ?30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca²⁺ spikes with a maximum rate of rise of 7.5 Vs^?1 in microstome and 4.7 Vs^?1 in macrostome cells. Depolarizing voltage steps from a holding potential of ?40 mV induced an inward Ca²⁺ ‐current (I_ca) peaking at ?10 mV, reaching approximately the same value in microstome (?1.4 nA) and macrostome cells (?1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I_Ca in these morphs supports the notion that the voltage‐gated Ca²⁺ channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na⁺‐free solution and was only partially inhibited by extracellular Cs⁺. The inward rectification was completely blocked by replacing Ca²⁺ with Co²⁺ or Ba²⁺ in the recording solution, and is probably due to Ca²⁺ ‐activated inward K⁺ current secondary to Ca²⁺ influx through channels activated by hyperpolarization.     

Morphology and ontogenesis of Platyophrya bromelicola nov. spec., a new macrostome-forming colpodid (Protists,Ciliophora) from tank bromeliads of Jamaica

Platyophrya bromelicola nov. spec. was discovered in tanks of bromeliads from Jamaica. Its morphology, ontogenesis, and small-subunit rDNA were studied using standard methods. Platyophrya bromelicola differs from its congeners mainly by the pyriform, unflattened body (vs. reniform and flattened); the free-swimming (planktonic) habit (vs. biofilm creepers); and the unique ability to form two distinct morphs, i.e., small, bacteriophagous microstomes and large, predaceous macrostomes. Microstomes and macrostomes can be distinguished not only by body size and feeding preferences but also by the postoral pseudomembrane composed of two vs. three to four dikinetids per kinety. The ability to form macrostomes is considered as an adaptation to the highly competitive habitat. Ontogenesis closely resembles that of other members of the family. Platyophrya bromelicola is distinct not only morphologically but also genetically (3.7% in the small-subunit rDNA) from P. vorax, a common, cosmopolitan moss and soil species.     

A Comparison of Cortical Proteins in Tetrahymena vorax Microstomes and Macrostomes1

Proteins of surface membranes and surface-related cytoskeletons in Tetrahymena vorax microstomes and macrostomes were compared by one-dimensional SDS polyacrylamide gel electrophoresis to see if protein differences could be detected that correlate with the transformation from one phenotype to the other. Some differences were observed. However, these alterations appear to result from the heat-shock procedure used to synchronize the microstome-to-macrostome transition. The apparent lack of transformationspecific changes in cortical proteins is discussed. Similarities and differences between cytoskeletal proteins of T. pyriformis GL-C and T. vorax are also noted and discussed.     

A silicone polymer as a Steroid Reservoir for enzyme-catalyzed Steroid reactions

Since steroids are only slightly soluble in the aqueous solutions in which enzymatic reactions take place, it is difficult to obtain high effective concentrations per unit reactor volume when enzymes are used to catalyze steroid reactions. In order to obtain high effective concentrations in the present work, we have used small particles of a hydrophobic polymer, poly (dimethyl siloxane), as a reservoir for the steroid substrate and product. The activity of a bacterial hydroxysteroid dehydrogenase in a buffer solution declines much more slowly in the presence of those polymer particles than in the presence of a comparable amount of butyl acetate or ethyl acetate, the organic solvents used as steroid reservoirs in previous work with steroid transforming enzymes. When another substrate of the hydroxysteroid dehydrogenase is loaded into the polymer particles and the particles are suspended in an aqueous solution containing the enzyme and its cofactor, more product is formed that when a similar solution is emulsified with butyl acetate.     

Studies of Macrostome Formation of Low-Transforming Tetrahymena vorax. Transformation Enhancers,Generation Time,and Membrane Fluidity1

ABSTRACT. Periodically, stocks of Tetrahymena vorax, which normally yield 70–90% macrostomes when subjected to heat shock or other induction methods, become low-transformers and yield ≥30% macrostomes. The addition to the post-heat-shock wash buffer (pH 6.8) of 2.7 × 10^-4 M Fe³⁺, 1.6 × 10^-5 M Cu²⁺, 1 × 10^-4 M retinol palmitate or the adjustment of the buffer to a pH of 4 to 5 boosts transformation significantly over controls in inorganic medium alone. The addition of Fe²⁺ or Cu¹⁺ has a similar, but less pronounced effect on transformation. Ferric ion (2.7 × 10^-4 M) will significantly increase transformation in starved non-heat-shocked cells, whereas Fe²⁺, copper, retinol palmitate, and hydrogen ion concentration have no effect. The agents that boost transformation appear to act by delaying cell division in pre-transformants. Membrane fluidity, as inferred by fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene, is altered in a consistent manner by starvation and heat shock. Enhancing agents, including compounds previously shown to boost heat-shock-induced macrostome formation, produce diverse shifts in membrane fluidity. Their effect on transformation of these low-transforming cells therefore appears to be attributable to some mechanism or mechanisms other than a direct alteration of membrane physical properties.     

Ortho-phthalaldehyde: a possible alternative to glutaraldehyde for high level disinfection

Ortho-phthalaldehyde (OPA) was tested against a range of organisms including glutaraldehyde-resistant mycobacteria, Bacillus subtilis spores and coat-defective spores. Glutaraldehyde (GTA) and peracetic acid (PAA) were tested for comparative purposes. Both suspension and carrier tests were performed using a range of concentrations and exposure times. All three biocides were very effective (> or = 5 log reduction) against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in suspension tests. OPA and GTA (PAA was not tested) were also very effective against Staph. aureus and Ps. aeruginosa in carrier tests. OPA showed good activity against the mycobacteria tested including the two GTA-resistant strains, but 0.5% w/v OPA was found not to be sporicidal. However, limited activity was found with higher concentrations and pH values. Coat-defective spores were more susceptible to OPA, suggesting that the coat may be responsible for this resistance. The findings of this study suggest that OPA is effective against GTA-resistant mycobacteria and that it is a viable alternative to GTA for high level disinfection.     

A stomatin dimer modulates the activity of acid-sensing ion channels

Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, we determined three crystal structures of the conserved stomatin domain of mouse stomatin that assembles into a banana-shaped dimer. We show that dimerization is crucial for the repression of acid-sensing ion channel 3 (ASIC3) activity. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 activity. In one crystal form, stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerization surfaces are also essential for the inhibition of ASIC3-mediated currents. The assembly mode of stomatin uncovered in this study might serve as a model to understand oligomerization processes of related membrane-remodelling proteins, such as flotillin and prohibitin.     

The Effect of the Lipogenic Inhibitor Cerulenin, on Macrostomal Cell Formation in Tetrahymena vorax

ABSTRACT. Macrostomal cell formation is blocked by the antibiotic cerulenin at levels of 15 μg/ml or higher. Inhibition can be reversed up to 4 h following cerulenin addition by washing and resuspending cells in new, noncerulenin-treated transforming principle. In these latter cases, additional time equal to the time spent in the inhibitor, is needed for cells to reach control values of transformation. Neither the addition of saturated or unsaturated fatty acids, cholesterol added alone or in combination with stearic acid, nor a mixture of lipids extracted from Tetrahymena vorax reversed the cerulenin effect. Radioisotope incorporation data showed while protein synthesis was reduced by the end of 1 h and tetrahymanol synthesis by the end of 2 h, little or no effect of this inhibitor occurred on RNA or fatty acid synthesis during these times. One interpretation of these results is that cerulenin, by preventing first protein synthesis and later tetrahymanol synthesis, interferes with synthesis and formation of membranes required for the microstome to macrostome transition.     

Effect of transition metals on stress, lipid peroxidation and antioxidant enzyme activities in tobacco cell cultures

to-baccoBright Yellow 2 (BY-2) suspension culture to understand the mechanisms of metal resistance in plant cells.We have analysed superoxide dismutase, catalase, and ascorbate peroxidase enzyme activities and superoxidedismutase-isoforms by isoelectric focusing gels in tobacco cells grown at two different toxic concentrations ofeach of the transition metals: copper, iron, manganese and zinc. Exposure of tobacco cells to these metals causedchanges in total superoxide dismutase activity in a different manner, depending on the metal assayed: after cop-perand manganese treatments, total superoxide dismutase activity was enhanced, while it was reduced after ironand zinc exposure. Superoxide dismutase-isoforms were affected by the metal used, and a Fe-SOD band with thesame isoelectric point as a Cu, Zn-SOD from non-treated cells, was induced after iron and zinc treatments. Cu,Zn-SODs were present in all metal-treatments whereas Mn-SOD was not detected in any case. Concerning otherantioxidant enzymes tested, such as catalase and ascorbate peroxidase, the latter showed a remarkable increase inactivity in response to copper treatments and catalase activity was enhanced after iron and with the lowest man-ganeseconcentration. Lipid peroxidation was increased after each metal treatment, as an indication of the oxi-dativedamage caused by metal concentration assayed in tobacco cells. These results suggest that an activation ofsome antioxidant enzymes in response to oxidative stress induced by transition metals is not enough to confertolerance to metal accumulation.     

The serine protease Esperase HPF inhibits the formation of multispecies biofilm

The antifouling (AF) potential of the serine protease Esperase HPF (subtilisin) was evaluated for the ability to prevent the formation of a four-species bacterial biofilm. The effects of enzyme activity, time and application of the enzyme were tested on the density and the oxidative metabolism of biofilm developed in microtiter wells. Esperase HPF did not inhibit the oxidative metabolism of the bacterial biofilm or planktonic growth, but the enzyme inhibited biofilm formation by its proteolytic activity as inactivated enzyme had no effect. The effective enzyme concentration was determined over a period of 72 h, as by then all the tested concentrations inhibited biofilm formation maximally. The effective concentrations of the enzymes in solution were the same regardless of time of application (ie before or after biofilm formation), but immobilisation of the enzymes caused a lower effective concentration. Esperase HPF is an attractive alternative to the biocidal compounds used in AF coatings today.     

Inhibition of leucocytic lysosomal enzymes by glycosaminoglycans in vitro.

1. A lysosomal fraction was separated by density-gradient centrifugation from a highly purified human polymorphonuclear leucocyte suspension. 2. Some 23 different lysosomal enzymes were assayed for activity in the presence of various concentrations of glycosaminoglycans. 3. The 21 acid hydrolases assayed were strongly inhibited to different degrees by low (0-12 mmol/l) concentrations of glycosaminoglycans in a pH-dependent manner. Thus inhibitions were stronger below pH4.5, with activity returning to control values at about pH5.0. 4. On a molar basis, the inhibitory activity for the several glycosaminoglycans studied was: heparin greater than chondroitin sulphate greater than hyaluronic acid. 5. Once the glycosaminoglycan-acid hydrolase complex was formed, it was partially dissociated by slight elevations in the pH of the incubation medium, by increasing the ionic strength of the incubation medium, or by adding several cationic proteins (e.g. histone, protamine). 6. As leucocytic lysosomes contain large amounts of chondroitin sulphate, and have a strongly acid intragranular pH, we suggest that glycosaminoglycans may modify lysosomal function through the formation of complexes with lysosomal enzymes, by inhibiting the digestive activity of the acid hydrolases when the intralysosomal pH is below their pI.