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At least 991 records · Page 55Linked to original sources
PubMed1985

Modulation of the digestive lysosomal system in Paramecium caudatum. II. Physiological effects of cytochalasin B, colchicine and trifluoperazine.

The heterophagic pathway of the digestive lysosomal system in Paramecium caudatum includes at least four steps: digestive vacuole (DV) formation, acidification-condensation, lysosomal fusion-digestion, and defecation. The second and the third require about 20 min, during which DVs are not egested. Because these steps occur at predictable intervals, the mechanism for each can be explored by exposing labeled DVs to different drugs prior to each step. In this study the effects of cytochalasin B (CB), colchicine and the calmodulin antagonist, trifluoperazine (TFP), were studied. All three drugs inhibited DV formation in a dose-dependent manner when cells were pulsed with latex beads and a drug simultaneously. TFP was cytotoxic above 5 microM. Vacuole formation was completely shut down when cells were pre-exposed to 5 microM TFP for 13 min. At this level, the duration of the acidification step was lengthened, and the rate of defecation decreased with increasing exposure. These results suggest that these inhibitory effects may be more related to TFP's cytotoxicity than to its action on calmodulin-mediated process. Colchicine at 1 mM had no effect on the third or fourth step, but inhibited the acidification step so that DVs were egested later and at a slower rate. Exerting a differential effect on all four steps, CB inhibited DV release from the cytopharynx, egestion of defecation-competent DVs at the cytoproct and lengthened the duration but did not block the lysosomal fusion-digestion step of the acidic DVs; it was most potent in blocking acidification, which prevented both lysosomal fusion with the labeled DVs as well as DV egestion, the latter for more than 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Rapid and sensitive assays for phagosomal acidification in Paramecium and Tetrahymena.

Biochemical and cytochemical procedures were developed to measure the rate of phagosomal acidification for phagosomal pH ranging from 5 to 2.5. These assays were based on the pH-dependent inactivation with time of horseradish peroxidase (HRP) activity, a result attributable to the dissociation of this enzyme to a colorless protein and ferriprotoporphyrin in acidic solutions. When preincubated in buffers of varying pH, the rate of HRP inactivation followed a sigmoid curve, with the highest rate of inactivation between 4.3 and 3.5 when using citrate-phosphate buffer and between pH 3.4 and 2.8 when using the universal ABC buffer. This inactivation was temperature but not concentration dependent. When Paramecium caudatum, members of the P. aurelia complex or Tetrahymena thermophila was pulsed briefly with HRP and small fluorescent beads, the loss of HRP activity, measured biochemically in cell homogenates and/or cytochemically in phagosomes, was rapid and followed the kinetics of a first-order rate reaction. Both assays gave similar values for the rate constant for acidification and similar rates of inhibition when P. caudatum was exposed to a proton ionophore, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. These assays can readily be adapted to other phagocytic cells as long as a rapid procedure is available for removing all unphagocytosed HRP and latex beads. These procedures are sensitive and rapid thus allowing many samples to be quickly prepared and analyzed.

Animals↗
PubMed1979

[Effect of local UV irradiation of the generative nuclei of Paramecium caudatum on the sexual functions of the cells].

A study has been made of the functional pattern of the micronucleus (Mi) during the sexual process in ciliates Paramecium caudatum which are descendants of those individuals whose Mi was locally irradiated with UV in dose of 3060 erg/mm2. It was demonstrated that these descendants (cells of UV-clones) were able to enter into the sexual process whatever morphological type of their Mi might be. In 4 of 14 tested clones, the initial mating type got changed for the opposite one, which may be associated with the Mi genome alteration following UV irradiation. The nuclear reorganization during the sexual process was studied in three UV irradiated clones. The local UV irradiated of the Mi is followed by the number of abnormalities in the derivates of Mi during the metagamic phase of the sexual process in clones whose Mi underwent serious changes. Exconjugants obtained from UV-clones with such changes in Mi and normal test-clones demonstrated a lower viability.

Animals↗
PubMed1979

[Effect of the spatial environment on the mitotic activity, size and protein content of Paramecium tetraurelia].

Cell proliferation, cell volume, protein components of paramecia and chemical analysis of culture medium were studied in Paramecium tetraurelia cultures placed in space environment. The space flight resulted in a marked stimulation of cell proliferation, an increase of cell volume, a decrease of mean protein components and changes of electrolytes in the culture medium.

Animals↗
PubMed1982

Differential distribution of voltage-dependent calcium channels and guanylate cyclase in the excitable ciliary membrane from Paramecium tetraurelia.

A novel method for isolation of cilia and ciliary membrane vesicles from Paramecium tetraurelia has been developed. Using a continuous Percoll gradient of low osmolarity after fragmentation of purified cilia by French Press treatment two membrane fractions with different buoyant densities were obtained. These fractions were further purified by conventional discontinuous sucrose density gradients and characterized biochemically and by electron microscopy. Guanylate cyclase, a membrane bound enzyme, was found almost exclusively in membrane vesicles of high buoyant density while the voltage-sensitive calcium-channel of the ciliary membrane was predominantly localized in low density vesicles. Examination of both fractions by SDS polyacrylamide gel electrophoresis revealed only minor differences in protein pattern in the 34 and 64 kilodaltons range. Morphologically both membrane vesicle fractions had a diameter of about 300 nm, however, the high density vesicle fraction contained a considerably larger amount of multilamellar structures with a multishell, onion-like appearance. Freeze-fracture analysis failed to detect differences in intramembrane particle content between low and high density vesicles. The possible biological relevance of the spatial separation of the calcium-sensor enzyme guanylate cyclase and the voltage-sensitive calcium-channels in the ciliary membrane is discussed in terms of a diffusion controlled mechanism for graded signal transmission.

Animals↗
PubMed1984

Characterizations of six ethanolamine sphingophospholipids from Paramecium cells and cilia.

Six ethanolamine sphingophospholipids from axenically cultured Paramecium tetraurelia were isolated from cells and purified ciliary fractions, and were characterized. The sphingolipids comprised 10.7% of whole cell and 32.5% of ciliary ethanolamine phospholipid fractions purified by ion exchange column chromatography. The individual sphingolipids were characterized by thin-layer chromatographic analyses of parent compounds and the polar head group and long chain base moieties, gas-liquid chromatography, and mass spectrometry of amide-linked fatty acids and long chain bases, and nuclear magnetic resonance of the compounds. Colorimetric assays of differential hydrolysis products and 31P nuclear magnetic resonance were used to determine the nature of phosphorus linkages. The sphingolipids were identified as N-acyl-trans-4-hydroxy-sphinganine-1- phosphonoethanolamine , N-acyl-trans-4-hydroxy-sphinganine-1-phosphoethanolamine, N-acyl-sphingenine-1- phosphonoethanolamine , N-acyl-sphingenine-1-phosphoethanolamine, N-acyl-sphinganine-1- phosphonoethanolamine and N-acyl-sphinganine-1-phosphoethanolamine. All six had greater than 90% saturated fatty acids. These sphingolipids were quantified by radioisotope methods and plate densitometry of thin-layer chromatograms. Changes in the relative amounts of each species were detected in cells grown in different culture media as well as in cells at different culture ages.

Animals↗
PubMed1981

Axenic Paramecium caudatum. III. Biochemical and physiological changes with culture age.

As Paramecium caudatum passes through the lag, log and stationary phases of the culture cycle, cellular protein content, polar (PL) and neutral (NL) lipid contents, marker enzyme activities, rate of digestive vacuole formation and cellular viability undergo characteristic changes. Maximal protein content (60 ng/cell) and enzyme activities ranging from 7 nmoles for catalase to 0.2 pmoles/cell/min for alkaline phosphatase were observed between days 2 and 4. This active metabolism paralleled the fine structure of 1 to 3 day-old cells which contained extensive foci of rough endoplasmic reticulum (RER) partially bordered by Golgi stacks and the rapid depletion of those lipid fields accumulated during day 1. Decrease in protein content and enzyme activities in late log phase indicated a slowing of cellular synthesis. The lipids in the medium were largely depleted and accounted for the low lipid uptake of 14 ng/cell on day 5 as compared with 615 ng/cell on day 1. Yet a vast amount of protein lysate was still available in the culture medium. During stationary phase, catalase activity remained constant, but activities of alkaline and acid phosphatases and 5'nucleotidase declined gradually to low levels, while those of Ca2+-ATPase and malate dehydrogenase declined precipitously. Only 25% of the maximal activities of the latter two enzymes remained by the end of stationary phase. A ten-fold increase in the cellular PL and NL content was already observed 24 h postinoculation. This accumulation was used for subsequent growth and cell divisions; PL declined exponentially and NL less steeply between days 1 and 6. PL remained level (5 ng/cell) throughout stationary phase while NL declined further to 1 ng/cell by day 11. The rate of digestive vacuole formation was constant (6.3 +/- 0.5 DV/5 min pulse) during the entire log phase, then declined from 4.4 on day 6 to 0.22 on day 11. When early to mid-stationary-phase cells were subcultured, some lag in growth was seen; a definite lag was observed when inoculating with late-stationary-phase cells. When early-death-phase cells were given fresh nutrients, many died; the surviving ones became fully rejuvenated after 48 h. The biochemical and physiological data from this study are correlated with the morphological study of the companion paper.

Alkaline Phosphatase↗
PubMed1981

Isolation of surface membranes from normal and exocytotic mutant strains of Paramecium tetraurelia. Ultrastructural and biochemical characterization.

A density gradient centrifugation method for the isolation of the surface membrane complex from Paramecium tetraurelia cells is presented. The resulting "pellicles" consist predominantly of the somatic cell membrane and the underlying alveolar membranes. Marker enzyme activities for other cell components are low and SDS-polyacrylamid-gel electrophoreses indicate the presence of only minor amounts of ciliary and secretory proteins. Pellicles were prepared from different strains: (a) Exocytosis-capable strains with the normal set of exocytotic organelles ("trichocysts") docked to the cell membrane (strains 7S, K 401, and 9-18 degrees C), (b) exocytosis-uncapable strains (although with normal trichocyst attachment: nd 9-27 degrees C, nd 6, nd 7) and (c) strain from tam 38 with empty docking sites and rare, defective, free trichocysts. A Ca2+-stimulated ATPase was present in the pellicles from all strains with Km (CA2+) values between 0.19 to 0.88 mM Ca2+ and Vmax between 286 to 787 nMoles Pi/mg protein/min. Km and Vmax was identical for all strains of group (a). Vmax was significantly lower for all strains of group (b) and still lower for group (c). Similar group differences were found for Km (except for strain nd 6). Freeze-fracture analysis shows that the disruption of the membrane-to-membrane attachments during fractionation is paralleled by the disarrangment of the regular arrays ("rings", "rosettes") of membrane-integrated particles.

Animals↗
PubMed1983

Effects of dimethylsulfoxide (DMSO) on the digestive-lysosomal system in Paramecium caudatum.

The effects of DMSO (dimethylsulfoxide) on cell growth and on the digestive-lysosomal system of axenically grown Paramecium caudatum were studied. A general protocol of exposing cells to different concentrations of DMSO at the beginning of each of the four processes in the digestive cycle enabled us to analyze the effect of DMSO at each step. Vacuole formation and the beginning of a digestive cycle were initiated by adding latex beads to the cells. Maximum cell densities at stationary phase of growth were found to be inversely proportional to DMSO between 0.5 and 1.75%, and the duration of the generation time was exponentially proportional. At 2% DMSO cellular division was completely blocked, and above 2% it was cytotoxic. P. caudatum survived for 8 h in 4% DMSO and died instantaneously in 10%. This inhibitory effect on growth was reversible, though this reversibility might depend on the duration and level of DMSO exposure. DMSO exerted a dose- and time-dependent inhibitory effect on the rate of DV formation but had little effect on the acidification-condensation and the lysosome fusion-digestion processes. The size of the DV formed was also reduced, and this effect was dose-but not time-dependent; vacuole size reduction occurred immediately with DMSO exposure, and no further reduction was observed during exposures of up to 24 h. DMSO at 3 and 4% inhibited vacuole defecation, but the cells could overcome this inhibition when exposed to DMSO for longer periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Conjugation and meiosis of Paramecium caudatum infected with the micronucleus-specific bacterium Holospora elegans.

Cells of two complementary mating types of Paramecium caudatum, syngen 3, are infected with the micronucleus-specific bacterium Holospora elegans. Cells with bacteria in their micronuclei show the mating reaction with agglutination and pair formation. All the stages of meiosis in this species are observed and pronuclei are formed. During the pregamic divisions the bacteria are distributed to the division products, but many of the bacteria remain within the separation spindles, later being released into the medium. Therefore, the pronuclei contain only a few or no bacteria. In a test of the viability of exconjugant clones CNR-mutant (a behavioral mutant) and wild type cells, both infected with H. elegans, are crossed. The survival of exconjugants is very low (10.6% compared to 57.3% in the control). After conjugation only parental types are found indicating the occurrence of macronuclear regeneration after conjugation. The same result is obtained with cells which had contained bacteria but were cured by means of antibiotics. It is concluded that infected micronuclei have become genetically defective and did not give rise of new functional macronuclei. Therefore, H. elegans-bearing paramecia are genetically dead, which shows the parasitic nature of the bacterium H. elegans.

Animals↗
PubMed1983

Phagosome fusion vesicles of paramecium. I. Thin-section morphology.

Ultrastructural studies of the digestive system of Paramecium caudatum focusing on the first 5 min of digestive-vacuole age reveal a set of vesicles, named phagosome fusion vesicles (PFVs), which fuse with the digestive vacuole just after the vacuoles are released from the cytopharynx and concomitant with vacuole acidification. Serial thin-sections of vacuoles labeled with horseradish peroxidase (HRP) and/or latex beads in pulse-chase studies were observed. PFVs, irregularly shaped, electron-translucent vesicles ranging from a small diameter to over 1 micro, are first seen in the region of the cytopharynx where they bind to the nascent vacuole membrane. Within 30 sec of vacuole release the PFVs fuse with the vacuole where they remain for a brief time connected to the vacuole by a narrow annulus. HRP-reaction product is found in vacuoles but not in PFVs before PFVs fuse with the vacuoles. After fusion with PFVs HRP is quickly inactivated. Tubular extensions of vacuole membrane then form between the fused PFVs. By 3 to 5 min both PFVs and tubules disappear from the vacuole surface and lysosomes appear in their place. We believe the tubules are pinched off as PFV membrane is being added to the vacuole. Microfilaments coat the membrane during all these dynamic events. Since the pH of the vacuole becomes acid during the first few minutes, we are now looking for a direct correlation between PFV fusion and acidification.

Animals↗
PubMed1982

[Comparative study of the proliferation of Paramecium tetraurelia aboard a satellite and aboard a stratospheric balloon].

A possible effect of cosmic rays on cell proliferation was investigated in cultures of Paramecium tetraurelia during a stratospheric balloon flight, with the techniques already used for the CYTOS experiments, performed aboard the orbital station Salyut 6. The results show that the stimulating effect of space on cell proliferation, reported in the CYTOS experiments, also occurs in the balloon flight. The respective roles of cosmic rays and weightlessness in the biological response are discussed.

Animals↗
PubMed1983

Phagosome fusion vesicles of Paramecium. II. Freeze-fracture evidence for membrane replacement.

Phagosome fusion vesicles (PFVs), a new population of relatively large granules in Paramecium caudatum which fuse with the first stage of digestive vacuoles (DV-I) shortly after these vacuoles are released from the cytopharynx (their site of formation), have been studied by using the freeze-fracture technique. Identification of PFVs is possible in the resulting replicas at all sites where they are commonly found in thin sections, at the cytopharynx, bound but not fused with nascent digestive vacuoles and fused with released vacuoles in the cell's posterior end. These PFVs have membranes which do not resemble the membranes of the forming digestive vacuole membrane or the discoidal vesicle membranes from which vacuole membrane is derived. Their smooth E-fracture face with only 50 to 100 intramembrane particles (IMPs) per micrometers 2 and particulate P-face (approximately 2500 IMPs/micrometers) do resemble the second vacuole stage (DV-II) which is characterized by a smaller diameter and acid pH. Evidence is presented for PFV fusion with the DV-I and for membrane replacement, at least in part, as the DV-I becomes a DV-II. Membrane replacement entails first adding PFVs to the DV-I and then removing the original discoidal vesicle-derived membrane as tubules as the vacuole condenses. Implications of the possible role of PFVs in forming intravacuolar symbiotic relationships are also discussed.

Animals↗
PubMed1978

Membranous sacs associated with cilia of Paramecium.

A membranous system associated with the cortical cilia of Paramecium putrinum is described. A sac lies below the cortical kinetosomes and extensions from it pass along the kinetodesmal fibre to lie near, or make contact with, the parsomal sac and also pass deep into the cytoplasm to pass close to a mitochondrion. Because of its widespread occurrence in this and other species it is suggested that the system plays a physiologically active role.

Animals↗
PubMed1982

Proliferation kinetics of paramecium tetraurelia in balloon-borne experiments.

Experiments were carried out to demonstrate the effect of cosmic radiation, at a balloon-flight ceiling of about 36,500 m (120,000 ft) on single-cell organism proliferation. Paramecium tetraurelia were placed in air-tight containers and maintained at 25 degrees +/- 0.1 degrees C. Cellular growth was determined by cell count, either after recovery or during the flight, by means of an automatic fixation device. Dosimetry was performed by a tissue equivalent proportional counter and was of about 0.5 mrad/h. Flight ceiling duration ranged from 48 min - 22 h. A secondary stimulating effect of growth rate, preceded by a temporary decrease, was observed after recovery. Because of the high bacterial concentration in the trans-Mediterranean flight culture medium, the temporary drop of the growth rate, due to the radiolysis products, disappears. We consider that the stimulating effect can be the result of enzymatic intracellular scavenging of radiolysis products generated in the cell.

Altitude↗
PubMed1981

Calcium receptor protein calmodulin isolated from cilia and cells of Paramecium tetraurelia.

A low molecular weight protein of about 17 000 as determined by SDS-polyacrylamide gel electrophoresis has been isolated from cilia and cell bodies, respectively, from Paramecium tetraurelia (wildtyp 51s). This protein has been identified as calmodulin by various properties previously ascribed to calmodulin from other vertebrate and invertebrate systems. These properties are heat stability, electrophoretic mobility and its ability to activate in the presence of calcium a calmodulin-dependent phosphodiesterase from pig brain. Calmodulin is present in rather high amounts in cell bodies (75 micrograms/g) and also in isolated cell-free cilia (up to 50 micrograms/g). Its presence in cilia suggests a role in the control of ciliary activity.

Animals↗
PubMed1981

Axenic Paramecium caudatum. II. Changes in fine structure with culture age.

The fine structure of axenically grown Paramecium caudatum undergoes characteristic changes which reflect the cell's nutritional and metabolic conditions. Cells in early log phase of growth contain large lipid accumulations and extensive foci of rough ER bordered by Golgi stacks, indicating an active metabolism. Lipid bodies and ER foci are reduced during mid log phase. By days 5 and 6 (late log phase) some rough ER and a few lipid bodies are found in the cortex while the endoplasm is filled with free ribosomes and small vesicles. Conjugating cells, observed at the onset of stationary phase (day 6), show an accumulation of secondary lysosomes concomitant with a lack of digestive vacuoles. Lipofuscin-like granules and autophagosomes appear on day 7 and increase in number throughout the stationary and death phases. Immature autophagosomes are smaller and are bounded by a "thin" membrane lacking a glycocalyx. Mature autophagosomes (autophagolysosomes) containing partially digested mitochondria have a "thicker" membrane with a glycocalyx, a characteristic of lysosomes. Fusion of lysosomes with autophagosomes is verified by the presence of the natural lysosomal marker, the paracrystalline sheets, in the autophagolysosomes. The residue remaining from the lipid fields on days 5 to 20 includes highly convoluted membrane-like patches with long non-membrane-bound cylinders. Concomitant with the decrease in the number of digestive vacuoles, lysosomes, autophagosomes and lipofuscin-like granules proliferate from day 7 and become the striking feature of death-phase cells. With increased culture age the cisternae of Golgi stacks become dilated and decrease in diameter, and the number of ribosomes on the ER at the cis-face of these stacks is reduced. Mitochondria sometimes increase in density, and some have multi-lamellar whorls extending from their outer membrane.

Cell Survival↗
PubMed1978

[Effect of local UV irradiation of the generative nucleus on the vegetative functions of the infusorian, Paramecium putrinum].

The influence of the generative nucleus on the vitality in three clones of Paramecium putrinum was studied. The selective functional inactivation of the micronucleus (MI) was achieved with a local ultraviolet microbeam. After irradiation of micronuclei these soon disappeared and the irradiated cells perished shortly afterwards. In the part of UV treated-subclones the macronucleus (MA) fragmentation was discovered. It is possible that the fragments of MA are some kind of functional replacement of the lost or damaged generative nucleus. That data obviously demonstrate the function of MI of P. putrinum during its vegetative growth.

Cell Nucleolus↗