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Blepharismins are photodynamic hypericin-like dianthrones produced as a variable pigment blend in Blepharisma ciliates and mostly studied in the Afro-Asiatic Blepharisma japonicum. The present work describes the bioactivity of pigments from the Brazilian Blepharisma sinuosum. Comparative analyses showed that the pigments from both species can trigger photo-induced modifications in phospholipids, but different redox properties and biological activities were assigned for each pigment blend. Stronger activities were detected for B. sinuosum pigments, with the lethal concentration LC50 10 × lower than B. japonicum pigments in light-irradiated tests against Bacillus cereus and less than half for treatments on the human HeLa tumor cells. HPLC showed B. sinuosum producing a simpler pigment blend, mostly with the blepharismin-C (~ 70%) and blepharismin-E (~ 30%) types. Each blepharismin engaged a specific dose-response profile on sensitive cells. The blepharismin-B and blepharismin-C were the most toxic pigments, showing LC50 ~ 2.5-3.0 µm and ~ 100 µm on B. cereus and HeLa cells, respectively, after illumination. Similarity clustering analysis compiling the bioactivity data revealed two groups of blepharismins: the most active, B and C, and the less active, A, D and E. The B. sinuosum pigment blend includes one representative of each clade. Functional and medical implications are discussed.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme that catalyses the oxidative cleavage of L-Trp. The ciliate Blepharisma stoltei has four IDO genes (IDO-I, -II, -III and -IV), which seem to have evolved via two sequential gene duplication events. Each IDO enzyme has a distinct enzymatic property, where IDO-III has a high affinity for L-Trp, whereas the affinity of the other three isoforms for L-Trp is low. IDO-I also exhibits a significant catalytic activity with another indole compound: 5-hydroxy- l -tryptophan (5-HTP). IDO-I is considered to be an enzyme that is involved in the biosynthesis of the 5-HTP-derived mating pheromone, gamone 2. By analysing a series of chimeric enzymes based on extant and predicted ancestral enzymes, we identified Asn131 in IDO-I and Glu132 in IDO-III as the key residues responsible for their high affinity for each specific substrate. These two residues were aligned in an identical position as the substrate-determining residue (SDR). Thus, the substrate affinity and specificity are regulated mostly by a single amino acid residue in the Blepharisma IDO-I and IDO-III enzymes.

Mating pheromones (gamone 1 and gamone 2) in the ciliate Blepharisma are biologically active substances that trigger sexual reproduction (conjugation) under starvation conditions. Gamone 1 is a glycoprotein secreted by type I cells, and gamone 2 is a tryptophan (Trp)-derivative compound secreted by type II cells. Both gamones stimulate complementary mating type cells to promote each gamone production and induce pair formation. To elucidate the biosynthetic pathway of gamone 2, we investigated the enzymes involved in the pathway and the specificity of the enzymes. An RNA-seq analysis revealed that Blepharisma stoltei (Heterotrichea) possesses four indoleamine 2,3-dioxygenase (IDO) genes showing distinct expression patterns. Along with results from real-time PCR, these findings demonstrated that each IDO gene has different expression patterns that depend on the cellular conditions. Expression of IDO-I was correlated with the intensity of gamone 2 expression, and the recombinant IDO-I protein showed catalytic activity for 5-hydroxy-L-Trp (5-HTP) but very weak activity for L-Trp. Our results indicate that IDO-I is an enzyme evolutionary specialized to gamone 2 production in Blepharisma, and that the biosynthetic pathway for gamone 2 uses 5-HTP as an intermediate.

The unicellular eukaryote, Blepharisma japonicum, is a light-sensitive ciliated protozoa. It possesses a photoreceptor pigment called blepharismin that plays critical roles in defensive behavior against predators and step-up photophobic response. In addition, the pigment generates reactive oxygen species such as singlet oxygen and hydroxyl radicals which contribute to photodynamic action. Previous studies reported that intense light (>300 W m−2) induced rapid photodynamic killing (necrosis) characterized by cell swelling and plasma efflux, while moderate light (3–30 W m−2) only induced pigment extrusion and photooxidation. We have found that moderate light (5 W m−2) induced apoptosis-like cell death. Microscopically it was found that >3 h of moderate light irradiation induced macronuclear condensation and plasma efflux without cell swelling. Single cell gel electrophoresis assay showed that DNA fragmentation occurred between 1 and 3 h of irradiation, and the condensed macronuclei contained quite fragmented DNA. Macronuclear DNA extracted from light-irradiated cells contained DNA fragments of 180–200 and 360–400 bp, which were seen as apoptosis ladders.

Ciliates communicate via diffusible signaling pheromones that have been identified in association with genetic mechanisms of mating types, either binary or multiple, that control the ciliate-specific sexual phenomenon of conjugation. Except for a glycoprotein and a tryptophan-related molecule acting as pheromones in Blepharisma, the other pheromones are cysteine-rich proteins varying in length among Euplotes species from 32 to 109 amino acid residues. These proteins are all structurally homologous sharing a common three-helix bundle fold, which allows them to bind cells in competition with one another in close analogy with the families of protein growth factors and cytokines of multicellular organisms. Although identified in the extracellular environment for their mating-induction activity, pheromones act also, and presumably primarily, as autocrine growth factors that bind to, and promote the reproduction of the same cells from which they are constitutively released.

As in animals and other multicellular organisms, protozoan ciliates communicate via diffusible signalling pheromones. These pheromones have been identified in the culture supernatant of various species. However, their isolation and definitive structural characterization have been achieved only in species of Blepharisma and Euplotes. With the exception of the two B. japonicum pheromones represented by a tryptophan-related molecule and a glycoprotein, all the other pheromones isolated from Euplotes species are proteins that vary in extension between 38 and 109 amino acids. They form species-specific families of structurally homologous members, in full accord with their multi-allelic determination at an apparently single genetic locus. The determination of the Nuclear Magnetic Resonance (NMR) solution structures of E. raikovi and E. nobilii pheromones has provided direct evidence that, although structurally unique, Euplotes pheromones adopt a common, species-specific three-helix folding. This close s...

— The toxic and phototoxic properties of blepharismin and oxyblepharismin that were purified from the pigments of a ciliated protozoan, Blepharisma japonicum, by thin-layer chromatography, were investigated in detail. The toxicity was tested against the ciliated protozoan Dileptus margaritifer, which is relatively sensitive to blepharismin. Although oxyblepharismin has been believed to be neither toxic nor phototoxic, it was found that oxyblepharismin is toxic in the dark and is also phototoxic. This shows that oxyblepharismin can act as a photosensitizer. The toxicity and phototoxicity of these pigments were compared with those of hypericin, which is known to be a typical, strong photosensitizer from plants. It was concluded that blepharismin and oxyblepharismin have strong intrinsic toxicities in the dark compared with hypericin, but their phototoxicities are slightly weaker than that of hypericin. This strong intrinsic toxicity supports our proposal that blepharismin acts as a defensive device against predators in the dark as well as in the light. The decrease in the defensive ability and the increase in the resistance to photokilling of Blepharisma concomitant with its color change from red to blue-purple in response to weak illumination can be explained by the decrease in toxicity and phototoxicity of the pigment itself and by the decrease in amount of the pigment.

The aim of the research presented here was to determine whether low concentrations (1 μg/ml) of a highly specific microtubule depolymerizing agent, nocodazole (NOC), affects the meiosis ac tivation process in conjugating pairs of the ciliated protozoan Blepharisma japonicum. In order to identify and quantify differen ces in meiosis, both NOC treated and untreated coeval pairs were stained and examined for their meiotic phases. A clear‐cut delay in the progression of meiosis in treated pairs was found. The findings indicate that NOC reversibly inhibits meiosis induction and that treated pairs need both continuous cell union and a period of recovery in order to progress through meiosis after NOC is with drawn. These results suggest that cytoplasmic microtubule dynamics may be involved in the meiotic activation processs of Blepharisma conjugating pairs.

The quinone pigment blepharismin isolated from Blepharisma, which is believed to be a photoreceptor pigment mediating photobehaviour (P. Scevoli, F. Bisi, G. Colombetti, F. Ghetti, F. Lenci and V. Passarelli, Photomotile responses of Blepharisma japonicum. I. Action spectra determination and time-resolved fluorescence of photoreceptor pigments, J. Photochem. Photobiol. B: Biol., 1 (1987) 75–84; T. Matsuoka, S. Matsuoka, Y. Yamaoka, T. Kuriu, Y. Watanabe, M. Takayanagi, Y. Kato and K. Taneda, Action spectra for step-up photophobic response in Blepharisma, J. Protozool., 39 (1992) 498–502), killed mammalian cells (L cells) when activated by light. Electron spin resonance (ESR) spectroscopy demonstrated that light-activated blepharismin generated hydroxyl radicals (.OH). Light-activated blepharismin pigment promoted lipid peroxidation of L cells, which was partially suppressed in the presence of the singlet oxygen quencher sodium azide (NaN3). The results suggest that the photodynamic action of blepharismin is correlated with lipid peroxidation which may be caused by hydroxyl radicals and/or singlet oxygen (1O2) produced via reactions sensitized by the pigment.

Blepharismin is an endogenous photosensitizing pigment found in the protozoan Blepharisma . This pigment inhibited the generation of Superoxide anion (O 2 − ) in neutrophils not only via a diacylglycerol-induced protein kinase C (PKC)-dependent reaction but also by an arachidonate-induced PKC-independent reaction. The inhibition was light and concentration dependent for both reactions. Light-activated inhibition was strong at wavelengths between 520 and 570nm but not above 610nm. PKC activity in neutrophils and from rat brain was inhibited by blepharismin in a light- and concentration dependent manner. Moreover, arachidonate-activated NADPH oxidase activity in a cell-free system was also inhibited by the pigment in a light- and concentration-dependent manner. These results suggest that blepharismin inhibits NADPH oxidase activation through the non-specific inhibition of various membrane-bound enzymes and that this inhibition may also be correlated with that of PKC.

The red pigment granule of Belpharisma japonicum is believed to be a photoreceptor organelle mediating photodispersal. Freeze-fracture and thin section electron microscopy revealed that the pigment granules contained a honeycomb-like structure constructed of folded membranes. In the fracture face of the honeycomb-like structure, small membrane particles were observed, which might correspond to pigment—protein complexes. The pigment granules were isolated and detergent-solubilized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the pigment granules mainly contained a 200 kDa membrane protein. The complex of red pigment and 200 kDa protein was isolated by gel-filtration chromatography of the detergent-solubilized components, and the protein was subjected to SDS-PAGE and amino acid analysis. The 200 kDa protein could not be dissociated into subunits by 2-mercaptoethanol, indicating that the protein is composed of a single polypeptide chain. Hydrophobic amino acids contained in the 200 kDa protein were not dominant, suggesting that only partial domains may extend across the membrane of the honeycomb-like structure.

Intracellular rearrangements of chloroplasts with respect to the direction of light, e.g. as found in Vaucheria, Adiantum, Mougeotia and Mesotaenium , are model systems for investigating the processing of light signals from perception through transduction to the terminal response. Two photosensory pigments, namely blue-/UV-absorbing pigment and — in a few exceptional species — the red/far-red reversible phytochrome system, are localized in the cytoplasm outside the chloroplasts. Dichroic orientation of the pigments is important for the detection of light direction via generation of an intracellular absorption gradient. Chloroplast movement, resulting in altered positioning, is based on the actomyosin system. Light-governed reorganization of the cortical and/or subcortical actin cytoskeleton results in biased chloroplast translocation. Besides polymerization or anchoring of actin to cell structures, the activity of myosin may be a point of control in some species. Local changes in membrane properties, including transient modulation of the membrane potential, appear to be the earliest steps in signal tranduction. The role of calcium and calmodulin, obviously involved in some species, is not clear as yet.

Blepharisma cells were attracted by a pellet of live bacteria (Enterobacter) which was separated from the Blepharisma suspension by a cellulose membrane (fractionation: M.W. 14,000). The cells, however, were not attracted by killed bacteria. Crude and heat-treated supernatants obtained from bacterial suspension also induced chemoaccumulation of cells. These results suggest that the cells of Blepharisma detect certain small molecules, produced by live bacteria, that can pass through the cellulose membrane and are stable to heat. From the live bacteria supernatant, several ninhydrin-positive substances were isolated by means of two-dimensional thin-layer chromatography. Several of the spots contained substances that attracted the cells, indicating that certain ninhydrin-positive components, such as peptides or free amino acids (probably products of bacterial metabolism), may serve as a signal for food.

The effect of the singlet oxygen quencher crocetin on the photomotile responses and photodynamic killing of the ciliated protozoan Blepharisma japonicum was investigated. The percentage of cells surviving photodynamic killing is higher in the presence of the water-soluble carotenoid derivative than in control samples. However, no significant difference is observed in the photobehavior of the control and crocetin-containing samples. These results indicate that blepharismin-sensitized photodynamic reactions occur in Blepharisma, but do not play any meaningful role in initiating photosensory processes.

Among many heterotrichous ciliates, Blepharisma japonicum especially demonstrates negative phototaxis in response to light stimulation, which is attributed to be caused by swimming acceleration accompanied by cell elongation and ciliary reversal. When Blepharisma cells were treated with 0.1 mM EGTA, cell elongation gradually decreased in its degree as the adaptation time lapsed. Calmodulin inhibitors such as W-7 (10(-5) M) and chlorpromazine (10(-5) M) inhibited cell elongation. These results suggest that a rise in cytoplasmic free Ca(2+) concentration might cause cell elongation through the Ca-calmodulin system. On the other hand, Ca(2+)-channel blockers (La(3+), Co(2+), Cd(2+), Zn(2+), Mn(2+) and verapamil) did not inhibit cell elongation. Ca2+ localization examined by calcium pyroantimonate cytochemistry suggests that Ca(2+) ions required for cell elongation might be supplied from the vacuoles located in the cortical region of the cell instead of Ca(2+) influx from the surrounding medium.

Time-gated fluorescence images of intact motile red and blue cells of the photoresponsive protozoan Blepharisma japonicum have been acquired, digitized and analyzed by means of a new dedicated micro-video system. The spatial and spectral maps of the intracellular distribution of the endogenous fluorophores reveal that red, as well as blue cells fluoresce mainly in the blue-green with a longer lifetime, whereas the vaculoes emit in the red with a shorter decay time. Upon breaking the cell membrane, the extruded pigments show a bright short-lived fluorescence in the red, attributed to blepharismin and oxyblepharismin in red and blue cells, respectively. The results on fluorescence time-behavior, apparent quantum yields and fluorescence spectral distribution are discussed with respect to the photosignalling function of blepharismin and oxyblepharismin.

An electrode sensitive to tetraphenyl phosphonium (TPP+) was applied to monitor the changes in the membrane potential of the ciliate Blepharisma japonicum during the process of cell elongation. Uptake of TPP+ by Blepharisma cells was observed when they were incubated in a medium containing 2 x 10(-6)M TPP+. Subsequent light stimulation (3,000 lux) led to an efflux of accumulated TPP+ from the cells, indicating that depolarization of the plasma membrane occurred with the concomitant cell elongation. TPP+ efflux was always observed whenever cell elongation was induced in response to various stimuli: valinomycin (10(-6)M), K+ (30 mM), Co2+ (5 mM), and Ca2+ (5 mM). Furthermore, elongation was also evoked by electrical stimulation. These observations indicate that membrane depolarization is involved in the process of cell elongation in Blepharisma.

— A computer-controlled apparatus for time-resolved laser fluorescence spectroscopy has been used to measure fluorescence lifetimes, time-integrated and time-gated spectra of crude extracts of blepharismin, the photoreceptor pigment of the ciliated photoresponsive protozoan Blepharisma japonicum, in ethanol, aqueous solutions and detergent micelles. The effect of hydroxyl concentration has been investigated in both alcohol and water solutions. A short-living (0.2-0.4 ns) molecular species, emitting at 600 nm, is predominant in aqueous solutions at pH < 11.7, whereas in pure ethanol solutions an intermediate-living species (about 1 ns), still fluorescing at 600 nm, prevails. Upon increasing OFF concentration, a third, long-living (about4–6 ns) molecular species, emitting at 660 nm, is formed in all the examined media. This species has been tentatively identified as the negatively charged form of the photoreceptor pigment, whereas the short-living and the intermediate-living fluorescence emissions have been attributed respectively to the phenolic and the quinonic neutral forms of blepharismin. The phenolic form in its ground state is suggested to be the molecular species from which proton release occurs.

Some functional derivatives of blepharismone, the low-molecular conjugation hormone of Blepharisma japonicum, were synthesised and tested for their biological activity. The lipophilicity constant of the substituent in 5-position of the aromatic system directly correlates with the conjugation-inducing effect of this substance. Any change in the N-formyl-position leads to compounds less active than blepharismone, non-active or even inhibitory. Blepharismone-related kynurenine derivatives are inhibitors of the gamone activity depending on structural relationship.

Eine leistungsfahige und variationsfahige Synthese von 3-(3-Indolyl)milchsaure-Derivaten bietet, unter anderem, einen guten Zugang zu rac-Blepharismon (7) und Analoga dieser Verbindung. Blepharismon induziert bei dem Ciliaten Blepharisma japonicum [fruher B. Intermedium (Bhandary)] die zum Kernaustausch fuhrende Konjugation. In Position 3 unsubstituierte Indole 1 werden mit rac-2,3-Epoxypropionsaure-methylester (2) zu den rac-3(3-Indolyl)milchsaure-methylestern alkyliert, die sich leicht zu den entsprechenden Sauren 4 verseifen lassen. Mit 5-Benzyloxyindol (1b) erhalt man rac3-(5-Hydroxy-3-indolyl)milchsaure (5), eine mogliche Vorstufe in der Blepharismon-Biosynthese. Spaltung des Pyrrolsystems durch Ozonolyse und Hydrogenolyse liefert die (Formamido)phenon-Derivate 6. Mit 1b erhalt man auf diesem Weg rac-Blepharismon (7) in 11proz. Gesamtausbeute. Preparation of 3-(3-Indolyl)lactic Acids,- A new synthesis of rac-Blepharismone, the Low Molecular Conjugation Gamone of Blepharisma japonicumA versatile and effective synthesis of rac-3-(3-indolyl)lactic acid derivatives offers - among other natural products of interest - good access to rac-blepharismone (7), the racemate of the conjugation inducing gamone of the ciliate Blepharisma japonicum [formerly B. intermedium (Bhandary)], and to analogues of this compund. Indoles 1, unsubstituted in position 3, are alkylated with methyl rac-2,3-epoxypropanoate (2) to give the methyl rac-3-(3-indolyl)lactates 3 which are easily hydrolyzed to yield the corresponding acids 4. rac-3-(5-Hydroxy-3-indolyl)lactic acid (5), the possible biogenetic precursor of blepharismone (7), is obtained from 5-benzyloxyindole (1b). Ozonolysis of the pyrrole system and hydrogenolysis lead to (formamido)phenone derivatives 6. rac-Blephraismone (7) has been produced in this way with 11% overall yield starting from 1b.

The light-induced avoiding reaction of Blepharisma Japonicum was investigated by means of microscope videorecording techniques. The percentage of photoreacting cells and the lag time between stimulus onset and reaction were measured as a function of the fluence rate and of the light stimulus wavelength. Action spectra indicate that the red pigment blepharismin is the photoreceptor pigment for this photoresponse. The fluorescence lifetimes and quantum yields of ethanol-extracted pigments and cold-extruded pigment granules were measured by means of a phase-modulation spectrofluorometer. The fluorescence lifetime and quantum yield of the pigment in granules are half those of pigments in ethanol solution. This could be in agreement with the hypothesis that blepharismin is the photoreceptor pigment for the observed photobehaviour.

The nucleotide sequences of 5S rRNAs from three ciliated protozoa, Paramecium tetraurelia, Tetrahymena thermophila and Blepharisma japonicum have been determined. All of them are 120 nucleotides long and the sequence of probable tRNA binding site of position 41-44 is GAAC which is characteristic of the plant 5S rRNAs. The sequence similarity percents are 87% (Paramecium/Tetrahymena), 86% (Paramecium/Blepharisma) and 79% (Tetrahymena/Blepharisma), suggesting a close relationship of these three ciliates.

SYNOPSIS. The macronucleus of the heterotrich ciliate Blepharisma undergoes a spectacular change in form in preparation for its division. The elongation phase of this cycle has been examined by light and electron microscopy. Coincident with elongation, microtubules appear closely applied to the outer surface of the macronucleus and parallel to the direction of elongation, and disappear as elongation is completed. It is demonstrated that the antimitotic drugs colchicine and podophyllotoxin reversibly block macronuclear elongation but do not entirely inhibit morphogenetic events including cell division. The failure of colchicine-treated macronuclei to begin or continue elongation is correlated with the prevention of formation, disruption, and/or disorientation of the extranuclear microtubules.

Local functional differences of the cell surface were investigated in the formation of cell unions in conjugation of Blepharisma intermedium . When cells of this ciliate are treated by the gamone (the conjugation-inducing substance) of the complementary mating type, they first unite by cilia and then more intimately by the direct contact of cell bodies. The ciliary union was found to be formed by a specific contact between two morphologically distinguishable surfaces, the adoral zone of membranelles (AZM) of one cell and the anterior extension of the undulating membrane (extUM) of the other. The AZM gains the capacity to unite first and the extUM next. The extUM of gamone-treated cells sticks not only to the AZM but also to glass and some other artificial substrata under some conditions. These stickings are inhibited by cycloheximide which also inhibits the ciliary union. The role of specific contact between AZM and extUM in the cell recognition is discussed.

The cyst walls of the ciliatesBlepharisma undulans andPseudomicrothorax dubius were examined ultrastructurally and by postembedding labeling with wheat germ agglutinin (WGA)-gold conjugate. Different methods of fixation and embedding were performed. In all procedures, WGA-gold binds selectively to material of the cyst wall. Pretreatment of the sections with chitinase inhibits labeling. The cyst walls of both species contain 3 nm fibrils, which are supposed to be of chitinous nature. In the cyst wall ofB. undulans, several thin layers of WGA-binding fibrils are interspaced with thick layers of other material. InP. dubius, WGA-binding sites are mainly concentrated in the mesocyst, where the microfibrils appear to represent the major component. These results obtained from two phylogenetically distant species confirm that chitin synthesis is an ancestral feature of ciliated protozoa. The amount and distribution of the chitin fibrils may play an important role in the properties and functions of the wall of the resting cyst.

Summary In a heterotrichous ciliate, Blepharisma japonicum , longitudinal elongation of the cell body is induced by light stimulation (1000 to 3000 lux). This light-induced response was inhibited under the existence of cyclic mononucleotide phosphodiesterase (PDE) antagonists such as papaverine (10 −4 M), theophylline (10 −3 M), dibutyril-cAMP (10 −4 M), 3-isobutyl-1-methyl-xanthine (10 −4 M) and dibutyril-cGMP (10 −4 M). Microinjection of cyclic mononucleotides, especially cGMP, inhibited cell elongation. These observations suggest that the cell elongation was mediated by intracellular cyclic mononucleotide. K + specific ionophore valinomycin (10 −8 –10 −7 M) enhanced light-induced cell elongation. This effect of valinomycin became more remarkable when valinomycin coexisted with PDE antagonists, while it was diminished under high K+ conditions. Moreover, the K + channel blockers tetraethylammonium (TEA) and CsCl inhibited cell elongation. These observations suggest that the cell elongation is also mediated by K + hyperpolarization, and that this electrical change is probably elicited after the intracellular concentration of cyclic mononucleotide decreased.

The macronuclear cycle in regenerating posterior fragments of albino Blepharisma intermedium consists of four sequential stages which result in the reversible condensation of the macronucleus, and which are similar to those in the pigmented strain. Tritiated deoxyuridine incorporation occurs predominantly during the first three hours of regeneration and then declines. Of the early hours, hour 2 exhibits a significant increase in the proportion of heavily-labeled cells. A statistical comparison of whole organisms labeled through exposure to radioactive precursor for a one-hour period with fragments labeled at each hour of regeneration reveals a significantly greater number of labeled fragments for each of hours 1 through 4. Asynchronous patterns of DNA replication are evident in the macronucleus of both regenerating and whole cells. Oval-shaped pellicular rings, consisting of dense accumulations of silver halide grains are evident during hours 5 and 6 of regeneration. Continuous inhibition of DNA synthesis during regeneration produces effects which depend upon the specific inhibitor employed. 5-Aminouracil (AU) effects an appreciable delay of regeneration, without lethal or abnormal consequences. AU, hydroxyurea (HU), 5 bromodcoxyuridine (BUdR) and 5-fluorodeoxyuridine (FUdR) exhibit a marked inhibitory effect upon division when they are administered to two-hour fragments. The first three hours of regeneration is the period of greatest susceptibility to inhibitor action with respect to post-regeneration division. AU, BUdR and FUdR interfere with the sequence of macronuclear events when these inhibitors are administered during hours 2–4 of regeneration. Contracted macronuclei are present at 24 hours and are related to division delay. HU, FUdR and BUdR treatment, during the later hours of regeneration, results in macronuclear fragmentation, branching and abnormal massing, respectively. Cytoplasmic abnormalities result from fragment exposure to the inhibitors during hours 4–6, with BUdR producing the most pronounced effects.

— –The intracellular pigment of the ciliate protozoan Blepharisma in the presence of oxygen sensitizes the cells to bright visible light (2700 foot candles (fc)). Illumination of the cells with dim visible light (200 fc) changes the color of the pigment to blue-gray; such cells are no longer sensitive to bright visible light. The pigment which exists in granules can be extruded by cold treatment and is slowly regenerated. The suspension of red cells, the extruded pigment from them and an ethanol extract of the pigment all have very similar absorption spectra. Illumination of the red pigment in ethanol converts it to the blue form of the pigment but only if oxygen is present, indicating a photooxidation. The pigment can be oxidized in darkness to the blue form by ozonation. A suspension of blue cells, the extruded pigment from them and an ethanol extract from them, all have similar absorption spectra. The pigments in red and blue form are very similar spectrophotometrically and in solubility in three species of Blepharisma studies: B. americanum, B. intermedium and B. japonicum. The purified pigment has strong absorption in the far (200–300 nm) ultraviolet (u.v.) and may serve as a screen against damaging U.V. radiation, especially as Blepharisma shows poor photoreactivation.

SYNOPSIS. An analysis of the pigments from the protozoan Blepharismn undtulans is reported. Methods of isolation and separation by paper chromatography are described. along with subsequent spectrophotometric and chemical analysis. Since the two major pigments were available only in amounts insufficient for thorough chemical analysis, these pigments were compared with the known pigment hypericin. Although similarities in the visible spectra indicate that the B. unduluns pigments possess the same mesodianthrone structure as hypericin, infrared spectra and chemical reactions disclose differences in the position, and possibly in the number, of the functional groups. A tentative structure is proposed.

SYNOPSIS. Autoradiographic studies on DNA-dependent RNA synthesis in isolated macronuclei of Blepharisma intermedium in vitro are described. Isolated macronuclei incubated in a medium containing RNA precursors incorporated cystidine and uridine into RNA. Uptake of cystidine was greater than that of uridine. Addition of ATP did not appreciably affect the uptake of precursors into RNA. Actinomycin D (5 μg/ml) blocked incorporation of cystidine and uridine. It is concluded that in isolated macronuclei of Blepharisma, RNA precursors are 1st converted into triphosphates and then incorporated into RNA thru DNA-dependent RNA polymerase.

SYNOPSIS. Cannibal giant Blepharisma undulans americanus, NYU strain, grown in mass culture, showed increased numbers of macronuclear lobes and greater variability in their number, compared to the bacteria-fed form. Feulgen and methyl greenpyronin preparations stained both the DNA and RNA of the cannibals and of the ingested forms within the food vacuoles. During the first 3 to 6 hours of digestion, macronucleus and deoxyribonucleic acid disappear from the food vacuole; during the last 6 hours the ribonucleic acid disappears from the food vacuole; DNA and RNA are therefore presumed to be digested and absorbed.

1. Blepharisma undulans, a protozoan with a reddish pigment, shows increased oxygen consumption under the influence of light. 2. If the light intensity is high, the animals are killed during a burst of oxygen consumption. 3. If the blepharismas are first bleached by exposure to light of low intensity they show only slightly increased oxygen consumption under the influence of light and they are not killed. 4. A preparation in which the animals are killed by heat still shows the increase in oxygen consumption on illumination with brilliant light. The supernatant solution does so as well, as does an alcohol extract of the dye. 5. The conclusion is drawn that the blepharismas are killed during photooxidation of the pigment, but the mechanism of action is not clear. Several possibilities are considered in the discussion.

Macromolecular syntheses were studied by incorporation of 14 C-labeled thymidine, 14 C-uridine and 14 C-amino acids, and by use of specific inhibitors of RNA, DNA, and protein synthesis upon the rate of regeneration of post-peristomial pieces of Blepharisma intermedium . The effect of these inhibitors on the macronuclear cycle which occurs during regeneration was also determined. Mitomycin, naladixic acid, and pheleomycin—inhibitors of DNA synthesis—if in relatively high concentration and applied for a long time, delayed regeneration. 14 C-Thymidine was incorporated into most fragments of unsynchronized cells slowly during regeneration, although it was incorporated actively into blepharismas just before division. Naladixic acid and phleomycin reduced 14 C-thymidine incorporation during regeneration. 14 C-Thymidine was not incorporated into macronuclei of cells cut soon after division (synchronized cells). 14 C-Uridine was incorporated actively immediately after removal of the peristome and continued throughout regeneration. The label at first was strongest in or over the macronucleus and near it; it was weak in the cytoplasm, becoming stronger with lapse of time after cutting. Actinomycin D, which retards regeneration most actively immediately after cutting, reduced incorporation of 14 C-uridine. 14 C-Histidine, 14 C-leucine and 14 C-amino acids (mixture) were incorporated into regenerating blepharismas. Puromycin retarded regeneration of Blepharisma most actively 1 to 2 h after cutting. Puromycin of the concentration which retards regeneration reduced the incorporation of 14 C-amino acids. The results suggest that cutting Blepharisma incites active RNA synthesis (mRNA?) in the macronucleus. The spread of RNA in the cytoplasm is accompanied by protein synthesis. The experiments suggest that macronuclear DNA synthesis is not essential to regeneration, but probably occurs when the cells are cut at the time they are in or near the synthetic stage.

SYNOPSIS. An initial rise in temperature increases the reproductive rate in Blepharisma intermedium; after it reaches the maximum, it gradually declines and finally fission is blocked. Cytochemical investigations made on Blepharisma acclimated to 28 C—the temperature at which the reproductive rate is maximum—show that conditions are optimum for cell division. Requisite quantities of glycogen, basic proteins, unsaturated lipids, enzymes alkaline and acid phosphatases are present. At 38 C the cell metabolism is altered, there is a decrease in the quantity of glycogen reserves, proteins and lipids, the enzyme activity diminishes, the free amino acid pattern changes and cell division is blocked. However, this block is transitory. Upon return to room temperature and when fresh culture medium is added, after a lag of 3 days, the cell metabolism becomes normal and the ciliates resume fission.

1. The respiration of Amoeba proteus was measured. 10 c. mm. of cells were found to use about 1.6 mm.3 of oxygen per hour at 20°C. The respiratory quotient was found to be nearly unity. 2. No anaerobic metabolism was found for Amoeba. 3. The respiration of Blepharisma was found to be from 3 to 7 mm.3 oxygen per hour for 10 mm.3 cells. The respiratory quotient was about 1. 4. Blepharisma was shown to have a definite anaerobic metabolism. 80 mm.3 cells caused the evolution of 12.5 mm.3 carbon dioxide per hour at 20°C. in the presence of bicarbonate.

FOR conjugation of Blepharisma intermedium to take place interaction of soluble gamones with the cells is essential for union between complementary mating types I and II (refs 1 and 2). Type I cells excrete gamone 1 which transforms type II cells so that they can unite. Type II cells excrete gamone 2 which similarly transforms type I cells. Moreover, each gamone promotes the production of the other. Gamone 1, blepharmone, is a glycoprotein of molecular weight 20,000 (refs 3 and 4). Gamone 2, blepharismone, is calcium 3-(2′-formylamino-5′-hydroxybenzoyl) lactate5.

Gamone 1 of Blepharisma intermedium was isolated, identified as a slightly basic glycoprotein (mizoleclular weight, 2 x 10(5)), and designated as blepharmnone. At the concentration of 6 x 10(-8) milligram per milliliter, it specifically transforms matinig type 2 cells, so that they can conjugate in about 2 hours.

Germ nuclei of Blepharisma japonicum begin meiosis within a few hours when cells of complementary mating types conjugate. We synchronized the onset of conjugation and treated cells in different stages of meiosis with 10 micrograms/ml cycloheximide which strongly inhibits protein synthesis in this ciliate. Cycloheximide arrested meiosis at six stages: I, between pairing of cells and swelling of germ nuclei; II, leptotene; III, zygotene; IV, pachytene; XI, interkinesis; XII, prometaphase II. Five of these arrests were reversible. Puromycin (250-500 micrograms/ml) also inhibited the progress of meiosis, though to lesser extents. We propose that the progression of meiosis of B. japonicum requires at least six proteins which are synthesized sequentially during meiosis.

Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.

Blepharisma (Figs. 1A and 1B) is a genus of ciliate protozoans in which most of the 47 listed species contain a pink pigment named blepharismin that renders them sensitive to glass-transmitted visible and nearultraviolet (UV) radiation. Initially, the pigment was called zoopurpurin by Arcichovskij (1905), who first determined its absorption spectrum. The name was changed to blepharismin to conform to the terminology of hypericin-like pigments, using the genus name as the basis of nomenclature: hypericin from Hypericum, stentorin from Stentor, fabrein from Fabrea, and blepharismin from Blepharisma (Giese, 1973).

SYNOPSIS. Observations made with tritiated thymidine uptake indicate that Blepharisma cannot utilize this precursor directly from the organic medium. An intermediary, such as a food organism (bacteria or Tetrahymena, is needed. These food organisms are not only capable of incorporating thymidine from the medium, but must also break it down or prepare it in a form that is utilizable for Blepharisma. DNA synthesis phase in Blepharisma occurs 17 hours after division. Maximum incorporation is obtained 18 and 19 hours postdivision and lasts thru 24 hours. This is followed by a 1–2 hour postsynthesis phase and division.

1. 1. Studies were made on sodium and potassium regulation in Blepharisma intermedium. Ionic levels were expressed in terms of μM/g protein. Sodium and potassium were measured with a Beckman DU spectrophotometer adapted for use as a flame photometer. Protein was measured by the Lowry method. 2. 2. The ionic levels of Blepharisma in different concentrations of a modified Taylor-Strickland balanced salt solution were studied. High internal potassium levels existed in all concentrations studied. Blepharisma maintained low internal sodium levels up to 20 × balanced salt solution. Above this concentration, internal sodium levels increased with external levels although the internal level was always maintained about 37 mM/1 below the environment. 3. 3. Blepharisma placed at 5 °C gain sodium and lose potassium in about equal amounts. Sodium is extruded and potassium accumulated when the animals are brought back to room temperature. Recovery in a K-free solution slows the rate of sodium extrusion. 4. 4. Inhibitors of oxidative metabolism (sodium azide, dinitrophenol, and sodium cyanide) and of glycolysis (sodium fluoride) produce an increase in internal sodium levels. Usually these inhibitors have little effect on the internal potassium levels but occasionally produce a slight, unexpected increase in internal potassium. 5. 5. Ouabain, an inhibitor of the sodium pump, produced a large increase in internal sodium and a small increase in internal potassium. 6. 6. Gum tragacanth and tetrodotoxin have little effect on sodium and potassium levels. 7. 7. Blepharisma was immobilized in salt solutions containing high sodium concentrations but not in solutions containing high potassium chloride, magnesium chloride, magnesium sulfate, calcium chloride, or sucrose. Sodium azide and dinitrophenol immobilize Blepharisma at low inhibitor concentrations. Other metabolic inhibitors immobilize Blepharisma at concentrations at which internal sodium has increased.