ATP und Ca2+ induzierte actomyosinartige Kontraktion glycerinisierter Makronuklei

Summary The addition of 2 mM-3 mM ATP to macronuclei ofParamecium bursaria suspended in a glycerol buffer medium causes a decrease in their volume up to 23% within 3 minutes. The infiltration medium must not only contain Ca²⁺, but must also be of low ionic strength for ATP to be effective. A slow, careful exchange of the glycerol medium for the contraction solution is also necessary. Ca²⁺ present alone in the standard contraction buffer can likewise induce a limited volume decrease; in the presence of low concentrations of Ca²⁺, Mg²⁺ shows no detectable effect on glycerinated nuclei. When the nuclear volume has been reduced by ATP in the presence of Ca²⁺, the addition of EGTA induces a reexpansion of the nuclei. Salyrgan, an organic mercurial, either prevents or abolishes the ATP-induced contraction. Other nucleotide triphosphates such as guanosine triphosphate (GTP), inosine triphosphate (ITP) or uridine triphosphate (UTP) likewise induce a volume decrease of the glycerinated macronuclei, but to a distinctly lesser extent than ATP.The results indicate that the volume decrease caused by the ATP-contraction solution is not a passive osmotic process. The resemblance to actomyosin contractions suggests that the volume decrease reported here might also be the result of the reaction of nuclear actomyosin and ATP.

     

Photosynthesis dependent acidification of perialgal vacuoles in theParamedum bursaria/Chlorella symbiosis: Visualization by monensin

Summary After treatment with the carboxylic ionophore monensin theChlorella containing perialgal vacuoles of the greenParamecium bursaria swell. TheParamecium cells remain motile at this concentration for at least one day. The swelling is only observed in illuminated cells and can be inhibited by DCMU. We assume that during photosynthesis the perialgal vacuoles are acidified and that monensin exchanges H⁺ ions against monovalent cations (here K⁺). In consequence the osmotic value of the vacuoles increases. The proton gradient is believed to drive the transport of maltose from the symbiont into the host. Another but light independent effect of the monensin treatment is the swelling of peripheral alveoles of the ciliates, likewise indicating that the alveolar membrane contains an active proton pump.Abbreviations HEPES N-(2-hydroxyethyl)piperazine-N-2-ethane sulfonic acid - DCMU 3-(3, 4-dichlorophenyl)-1,1-dimethylurea     

Structural comparisons between the soluble and the GPI-anchored forms of the Paramecium temperature-specific 156G surface antigen.

Biosynthetic labelling experiments performed on P primaurelia strain 156, expressing the temperature-specific G surface antigen, 156G SAg, demonstrated that the purified 156G SAg contained the components characteristic of a GPI-anchor. [3H]ethanolamine, [3H]myo-inositol, [32P]phosphoric acid and [3H]myristic acid could all be incorporated into the surface antigen. Myristic acid labelling was lost after treatment in vitro with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). After complete digestion by pronase, a fragment containing the intact GPI-anchor of 156G surface antigen was isolated. This fragment was shown to be hydrophobic and glycosylated and to possess an epitope found specifically in the GPI component of GPI-anchored proteins. The role of the GPI-tail in anchoring the 156G surface antigen into the membrane was assessed by determining that purified 156G molecules with the GPI-anchor could be incorporated into lipid vesicles and red cell ghosts whereas the 156G molecules lacking the GPI-anchor, as result of treatment with B thuringiensis PI-PLC, could not. It has also been shown that the membrane-bound form and the soluble form, obtained after cleavage of the 156G SAg lipid moiety either by an endogenous PI-PLC or by a bacterial PI-PLC, displayed identical circular dichroic spectra.     

Zur Taxonomie einer auxotrophen Chlorella aus Paramecium bursaria Ehrbg

Zusammenfassung Auf Grund der physiologischen Merkmale einer in Paramecium bursaria Ehrbg. auftretenden Chlorella ergibt sich eine systematische Zuordnung in den Formenkreis um Chlorella vulgaris f. tertia Fott et Nováková und Chlorella vulgaris var. vulgaris Beijerinck. Hiervon abweichende Befunde anderer Autoren werden diskutiert.

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On the taxonomy of an auxotrophic Chlorella isolated from Paramecium bursaria ehrbg

An auxotrophic Chlorella has been isolated from Paramecium bursaria Ehrbg. and cultivated in mass culture in an inorganic medium supplied with vitamins B₁ and B₁₂. With regard to its physiological properties it is not identical with either one of the so far known Chlorella species. It belongs, however, to the group of Chlorella vulgaris f. tertia Fott et Novaková and Chlorella vulgaris var. vulgaris Beijerinck.

     

Subcellular Effects of Cytochalasin B and Dimethylsulfoxide on Paramecium aurelia*

SYNOPSIS. Paramecium aurelia syngen 4, stock 57 (sensitive) cultivated in Cerophyl infusion were exposed to cytochalasin B CB and dimethylsulfoxide (DMSO), the solvent for CB, to distinguish between the effects of these agents on a cellular system. DMSO significantly inhibited survival, fission rate, [³H]leucine incorporation, and cell size. CB-treated cells generally had slower division and poorer survival rates than cells exposed to the equivalent DMSO concentration, although the [3H]leucine incorporation was generally greater at the lower CB concentrations than for DMSO alone. As seen by electron microscopy and a new grycerination technic for observing polysomes, DMSO caused nuclear (nucleolar, chromatin) abnormalities as well as membrane degradation and polysomal breakdown; CB caused the formation of aberrant membrane structures and ribosomal tetramers, crystals, and tubes.     

Localization of Possible Calcium-Binding Sites in the Cilia of Paramecium caudatum

SYNOPSIS. Electron-dense deposits indicating possible Ca-binding sites were found at the ciliary base of Paramecium caudatum fixed in a glutaraldehyde solution containing 5 mM CaCl₂. The deposits appeared mainly at the inner surface of the ciliary membrane above the "ciliary necklace" region, although they could also occur in the space between the outer and the central microtubules. In some cases a ring of exactly 9 deposits was found in a ciliary cross section of a cilium.     

Proteins and Polysaccharides In the Nutrition of Paramecium Multimicronucleatun

SYNOPSIS. Paramecium multimicronucleatum has been cultured for 20 years on a medium of salts, vitamins, amino acids, fatty acids, ribosides, and stigmasterol plus a little nondialyzable fraction (NDF) of baker's yeast. Fractionations of NDF identified 2 essentials: (a) in a fraction < 100,000 daltons which contained much protein and replaceable by ovalbumin and (b) in a fraction of < 300,000 daltons; this fraction contained much polysaccharide, replaceable by glycogen, which is > 300,000 daltons. For 2 years now P. multimicronucleatum has grown well with ovalbumin and glycogen replacing NDF. Besides ovalbumin, concanavalin A satisfies the protein requirement; this lectin attaches to sugar residues in glycogen. Studies with a fluorescent dye, PGA-1A, a stilbene derivative, provides further evidence for the polysaccharide requirement. This dye attaches to polysaccharides; when added to glycogen, and this in turn is added to a culture containing ovalbumin, fluorescent blue vacuoles appear within 2–3 h. When dye + glycogen were added to a culture without ovalbumin, no fluorescent vacuoles were found. A protein appears involved in formation of food vacuoles; this fits the pattern for endocytosis described in recent reviews. Besides glycogen, mannan gave good growth. Dextrin and amylopectin gave only fair growth through 7 serial transfers; glucose, maltose and amylose did not sustain growth. Strain 51 of P. tetratrelia , which grows well in NDF medium, grows well when NDF is replaced with ovalbumin and glycogen.     

Regulation of Macronuclear DNA Content in Paramecium tetraurelia*†

Synopsis.
The amitotic division of the macronucleus of Paramecium tetraurelia produces daughter macronuclei which frequently differ in DNA content. In wild-type cells these differences are small, but can be increased substantially by the action of mutant genes. The variance in macronuclear DNA content would increase continuously if there were no mechanism to regulate it. Paramecium has a very effective regulatory mechanism—all cells synthesize similar amounts of macronuclear DNA, regardless of the number of macronuclei or their prereplication DNA content. DNA synthesis is controlled at the level of macronuclear subunits, and the postreplication macronucleus consists of a mosaic of subunits that have undergone different numbers of replication events during the previous cell cycle. It is evident from experimental results that the amount of DNA synthesized can be influenced by the total size or mass of the cell. Experimental modification of the initial DNA content leads to no change in the amount of DNA synthesized, or in the subsequent protein content of the cells, but modification of cell size causes corresponding changes in the amount of DNA synthesized and in the size of the macronucleus. The implications of these observations for cell growth and the cell cycle are discussed.     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliates can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.