Evaluation of extraction procedures for the ion chromatographic determination of arsenic species in plant materials.

The determination of arsenic species in plants grown on contaminated sediments and soils is important in order to understand the uptake, transfer and accumulation processes of arsenic. For the separation and detection of arsenic species, hyphenated techniques can be applied successfully in many cases. A lack of investigations exists in the handling (e.g., sampling, pre-treatment and extraction) of redox- and chemically labile arsenic species prior to analysis. This paper presents an application of pressurized liquid extraction (PLE) using water as the solvent for the effective extraction of arsenic species from freshly harvested plants. The method was optimized with respect to extraction time, number of extraction steps and temperature. The thermal stability of the inorganic and organic arsenic species under PLE conditions (60-180 degrees C) was tested. The adaptation of the proposed extraction method to freeze-dried, fine-grained material was limited because of the insufficient reproducibility in some cases.

Determination of arsenic species in water, soils and plants.

Ion chromatographic separation coupled with ICP-MS was used to determine arsenic species in plant and soil extracts. A scheme for growth, harvesting, sample pre-treatment and analysis was developed for the arsenic species to enable determination. Preliminary results obtained with ten herb plants grown on arsenic-contaminated soil compared to non-contaminated soil show a heterogeneous pattern of accumulation rate, metabolization and detoxification mechanisms in monocots and dicots. Arsenite appears to be the major component in plants with good growth. Organic arsenic species were even detected at very low concentrations (< 150 microg kg(-1) (dry mass)).

Ultrastructural studies on a chlorella virus from Germany.

This is the first report on the morphology and fine structure of chlorella virus Göttingen-1 (CVG-1), an European member of the newly approved family Phycodnaviridae, which infects certain unicellular, eukaryotic, exsymbiotic, green algae. CVG-1 are polyhedral particles 145-160 nm in diameter. The capsid consists of two shells, apparently composed of ca. 7 nm subunits. Whereas the outer shell of the capsid appears to be icosahedral, the inner shell appears to be irregular underneath one vertex of the virion. In this vertex the inner shell is separated from the outer shell leaving a distinct space ("empty vertex") of unknown function.

A comparison of viruses infecting two different Chlorella-like green algae.

Five plaque-forming viruses (Pbi viruses) of the unicellular, eukaryotic, exsymbiotic Chlorella-like green alga strain Pbi were isolated from fresh water collected in Germany. The viruses were compared to two previously characterized plaque-forming viruses (NC64A viruses) of Chlorella strain NC64A. The Pbi viruses do not infect Chlorella NC64A and vice versa. Like the NC64A viruses the Pbi viruses are large polyhedron with a diameter of 140 to 150 nm, are chloroform sensitive, have many structural proteins, and have large dsDNA genomes of at least 300 kb. However, the Pbi viruses are serologically distinct from the NC64A viruses. The five Pbi virus genomes contain 5-methylcytosine, which varied from 14.2 to 43.1% of the cytosine, and two of them also contained N6-methyladenine. DNAs from the Pbi viruses hybridized poorly with the two NC64A virus DNAs and they have a higher guanine plus cytosine content (ca. 46%) than the NC64A virus DNAs (ca. 40%).

Comparative freeze-fracture study of perialgal and digestive vacuoles in Paramecium bursaria.

In the endosymbiotic unit of Paramecium bursaria (Ciliata) and Chlorella sp. (Chlorophyceae) algae are enclosed individually in perialgal vacuoles, which do not show acid phosphatase activity and thus differ from digestive vacuoles. Both types of vacuoles have been studied by freeze-fracture. Perialgal vacuoles are nearly spherical; their membrane always fits tightly to the algal surface. The vacuole size and shape do not vary much. During division of the algal cell into four autospores the vacuole diameter only doubles. After autospore formation the vacuole invaginates around the algal daughter cells and divides. Newly formed perialgal vacuoles remain in intimate contact and exhibit characteristic attachment zones before final separation. The two fracture faces of perialgal vacuole membranes are homogeneously covered with intramembranous particles (IMPs) but rarely show signs of vesicles pinching off or fusing with the membrane, except during vacuole division. The P-faces bear more IMPs (3164 +/- 625 IMP/micron 2) than the E-faces (654 +/- 208 IMP/micron 2). The range of IMP density on both faces is enormous, suggesting that the membrane is not static. Membrane changes are supposed to occur simultaneously with the enlargement of the vacuole and to be caused by fusion with cytoplasmic vesicles, as the fractured necks on vacuole membranes may indicate. Digestive vacuoles in P. bursaria show significant variations in size, shape, membrane topography and IMP density, as well as signs of endocytic activity. Different vacuole populations are present in P. bursaria according to different feeding conditions: ciliates fed for a long time have small vacuoles with few IMPs (322 +/- 198 IMP/micron 2 on the E-faces, 1438 +/- 458 IMP/micron 2 on the P-faces), which are probably condensed digestive vacuoles, whereas organisms fed for a short time have larger vacuoles with highly particulate faces (680 +/- 282 IMP/micron 2 on the E-faces, 2701 +/- 503 IMP/micron 2 on the P-faces) and thus are supposed to be older vacuoles. The digestive vacuole membrane changes continuously. Compared to digestive vacuoles perialgal vacuoles are characterized by small size combined with high IMP density on the two fracture faces. Their IMP densities resemble those of old digestive vacuole membranes. However, it would be premature to conclude that membranes of perialgal and old digestive vacuoles are identical. Membranes of old digestive vacuoles are mainly derived from lysosomal material, which presumably does not contribute to the formation of perialgal vacuole membranes as is indicated by the small vacuole diameter; fusion with lysosomes would considerably enlarge it.(ABSTRACT TRUNCATED AT 400 WORDS)

Participation of algal surface structures in the cell recognition process during infection of aposymbiotic Paramecium bursaria with symbiotic chlorellae.

The endosymbiotic unit green Paramecium shows a strong specificity of its partners. The aposymbiotic Paramecium bursaria forms a stable symbiotic unit only with a special strain of Chlorella sp. Algae suitable for symbiosis formation are enclosed in individual perialgal vacuoles whereas unsuitable algae are sequestered into food vacuoles. It is probable that algae are recognized by the ciliate because of specific surface structures rather than by their physiological properties. Experiments with synchronized algae demonstrate that autospores are taken up into perialgal vacuoles to a higher degree than mother cells, which have a different surface structure as shown by immunological techniques. Symbiotic algae treated with cellulase and pectinase or having been coated with specific antibodies or with lectins (concanavalin A or Ricinus communis agglutinin) are usually not recognized as suitable and are mostly sequestered into food vacuoles although they show the same physiological properties as untreated algae. These results indicate the participation of carbohydrate structures at the recognition sites of symbiotic chlorellae in Paramecium bursaria which interact during infection with special receptor molecules in the membrane of the ingestion vacuole of the ciliate.

Photobehaviour of Paramecium bursaria infected with different symbiotic and aposymbiotic species of Chlorella.

The endosymbiotic unit of Paramecium bursaria and Chlorella spec. shows two types of photobehaviour: 1) A step-up photophobic response which possibly depends on photosensitive agents in the ciliate cell itself - as is also shown by alga-free Paramecium bursaria - and can be drastically enhanced by photosynthetic activity of symbiotic algae; and 2) a step-down photophobic response. The step-down response leads to photoaccumulation of green paramecia. Both types of photobehaviour in Paramecium bursaria do not depend on any special kind of algal partners: The infection of alga-free Paramecium bursaria with different Chlorella species results in new ciliatealgae-associations. They are formed not only by combination of the original symbiotic algae with their host, but also by infection with other symbiotic or free-living (aposymbiotic) chlorellae, respecitively. Systems with other than the original algae are not permanently stable - algae are lost under stress conditions - but show the same types of photobehaviour. Photoaccumulation in general requires algal photosynthesis and occurs only with ciliates containing more than fifty algae/cell. It is not mediated by a chemotactic response to oxygen in the medium, since it occurs at light fluence rates not sufficient for a release of oxygen by the symbiotic system, e.g., below its photosynthetic compensation point. Photoresponses can be inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). Sensory transduction does not depend on any special symbiotic features of the algae, e.g., sugar excretion. The participation of oxygen in the Paramecium cell, of its cytoplasmic pH and of ions released or taken up by endosymbiotic algae in sensory transduction is discussed.

Evidence of de novo synthesis of maltose excreted by the endosymbiotic Chlorella from Paramecium bursaria.

The endosymbiotic Chlorella sp. from Paramecium bursaria excretes maltose both in the light and in the dark. Experiments on photosynthetic (14)CO2 fixation and (14)CO2 pulse-chase experiments show that maltose is synthesized in the light directly from compounds of the Calvin cycle, whereas in the dark it results from starch degradation.

The role of endosymbiotic algae in photoaccumulation of green Paramecium bursaria.

The endosymbiotic unit of Paramecium bursaria with Chlorella sp. photoaccumulates in white, blue-green, and red light (λ<700 nm), whereas alga-free Paramecia never do. The intensity of photoaccumulation depends on both the light fluence rate and the size of the symbiotic algal population. Photoaccumulation can be stopped completely with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport. Hence the photosynthetic pigments of the algae act as receptors of the light stimulus for photomovement and a close connection must exist between photosynthesis of the algae and ciliary beating of the Paramecium.

[The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosis. II. Symbiosis-specific properties of the physiology and the cytology of the symbiotic unit and their regulation (author's transl)]. / Die stoffwechselphysiologischen Beziehungen swischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose. II. Symbiose-spezifische Merkmale der Stoffwechselphysiologie und der Cytologie des Symbioseverbandes und ihre Regulation

The endosymbiotic association of Paramecium bursaria Ehrbg. with Chlorella spec. (green Paramecium) was studied both physiologically and cytologically. Comparison of the properties of the symbiotic unit with those of the symbiotic partners which had been isolated from it revealed the following features and differences: 1. Up to 6000 lux the photosynthetic capacity of the symbiotic unit is higher than that of the isolated symbiotic algae grown independently in mass culture under defined conditions. Alga-free Paramecium bursaria (colourless Paramecium) show a very low rate of CO2-fixation. 2. The green Paramecium has a higher compensationpoint of photosynthesis (4000-5000 lux) than the isolated alga (200-400 lux). 3. Green paramecia consume less oxygen in darkness than colourless organisms but more than the isolated algae. 4. The uptake of carbohydrates from the culture medium by green parpmecia is lower than the uptake by alga-free P. bursaria but higher than the one of the isolated algae. 5. Symbiotic algae within the intact symbiotic unit show tightly packed photosynthetic membranes and an intense disposition of starch. In the presence of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) or in darkness the arrangement of thylakoids is less compact and the deposition of starch is reduced. The growth and the number of the symbiotic algae in situ is regulated by a complex mechanism to which the intracellular level of carbohydrates belongs. The results are discussed in connection with ecological aspects of the Paramecium bursaria-endosymbiosis.

[The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosis. I. The nitrogen and the carbon metabolism (author's transl)]. / Die stoffwechselphysiologischen Beziehungen zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose. I. Der Stickstoff- und der Kohlenstoff-Stoffwechsel

Symbiotic Chlorellae have been isolated from Paramecium bursaria Ehrbg. and cultivated under conditions of nitrogen deficiency. Reinfection of Chlorella-free Paramecium bursaria with these nitrogen-deficient algae resulted in a complete regeneration and multiplication of the algae within the host cells. The endosymbiotic algal cells of the Paramecium bursaria-symbiosis can be supplied by their host with nitrogen. The inhibition of photosynthesis by 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) leads in green Paramecium bursaria to a breakdown of the symbiotic steady state-system resulting in a loss of algal cells. Obviously the endosymbiotic algae cannot be fed heterotrophically by their host to such an extent that a stable symbiosis is maintained. The application of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) can be used as a new method for culturing Chlorella-free Paramecium bursaria.