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[Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids].

Effect of glutamine and its metabolites (amino acids) on Chlorella glutamine synthetase (GS) (E.C.6.3.1.2) in the presence of Mg or Mn was studied. Purified GS preparation was used, isolated from Chlorella grown in the presence of NH as a sole nitrogen source. Glutamate, aspartate, alanine and glycine inhibit GS activity in the presence of both Mg and Mn. Tryptophane and valine (up to 15 mM) activate GS in the presence of Mn. Tryptophane inhibits GS in the system with Mg. Sinergistic inhibition was observed under the combined effect of amino acids on GS in the presence of Mn and aspartate or alanine. The change of GS activity observed is supposed to be due to the inhibitory effect of glutamine and amino acids studied, since the glutamine content is increased (in 2.5 times for 5 min) and that of alanine and dicarbonic amino acids (for the following 15 min) under NH assimilation in Chlorella cells.

Amino Acids

Experiments on the accumulation of lindane (gamma-BHC) by the primary producers Chlorella spec. and Chlorella pyrenoidosa.

Experiments were performed on the accumulation of the pesticide lindane (gamma-isomer of BHC) by two algae with different surfaces. An analytical procedure was developed for the gas chromatographic determinnation of lindane. At room temperature, lindane had a water solubility of 7.8 mg/L in distilled water and 6.7 mg/L in tap water. Under the experimental conditions of 10 to 100 microgram/L, 2.3% of the dissolved lindane was lost through adsorption on the glass walls of the equipment and 0.2% through evaporation. The recovery rate of lindane was 98% for the water samples and more than 90% for Chlorella spec. The tolerance in the gas chromatographic measurements amounted to 1.2%. Investigations on the effect of lindane on the growth of Chlorella spec. revealed irreparable damage to the algae cells through loss of chlorophyll, coagulation, and complete sedimentation at concentrations greater than 300 microgram/L. The experiments on sublethal accumulation showed the development of a state of equilibrium between the amount of lindane per cell and in the surrounding water with lindane concentrations of 10 to 100 microgram/L. The lindane was adsorptively attached to the algal cells within a few hr, and after three days lindane stabilized in the cells. The gelatinous surface of the algae increases the accumulation of lindane.

Chlorella

Detoxification of chlordecone poisoned rats with chlorella and chlorella derived sporopollenin.

Chlorella protothecoides accelerated the detoxification of chlordecone poisoned rats, decreasing the half-life of the toxin from 40 to 19 days. The ingested algae passed through the gastrointestinal tract unharmed , interrupted the enteric recirculation of the persistent insecticide, and subsequently eliminated the bound chlordecone with the feces. The detoxification was similar to that obtained with cholestyramine. Laboratory preparations were made to determine whether cell-free components retained the therapeutic properties of the whole cells. Acid and alkaline hydrolysis of the algae destroyed the cells except for the resistant cell wall components. One component was sporopollenin , a carotenoid polymer of limited natural occurrence among microorganisms and plants. Plant sporopollenin was not active, but algal cell walls and sporopollenin retained the therapeutic activity of the whole cells. The cells and cell walls have potential as detoxifying drugs for animals poisoned by chlordecone and other xenobiotic compounds with similar properties.

Animals

[Absorption of radiocobalt in Chlorella vulgaris]?1Assorbimento di radiocobalto in "Chlorella vulgaris".

The uptake, accumulation and loss of traces of radiocobalt present in the culture medium by a green alga, Chlorella vulgaris, were studied. The trace quantities of the radionuclide employed had no effect on the growth of the algal population. The uptake is related to the number of cells present and to the temperature of the culture medium; it is not affected by mitotic activity. The rate of the 60Co, uptake during algal growth is compared to the rate of uptake of other radioisotopes; 90Sr and, to some extent, 137Cs have the same rate of uptake as 60Co, whereas 90Y and 144Ce differ. Perhaps the radiocobalt in the cells undergoes a chelating process and consequently its loss to the outer environment is very slow. The involvement of a biological process, in addition to a physical one, is claimed in order to explain the possibility of an active concentration of the radioisotope (C.F.=170). Emphasis is laid on the possible danger of this concentration when a disposal system by dilution of industrial or nuclear waste is employed.

Cesium Radioisotopes

Correlations between characteristics of some free-living Chlorella sp. and their ability to form stable symbioses with Hydra viridis.

Aposymbiotic polyps of Hydra viridis were infected with 17 strains of in vitro cultured Chlorella sp. Larvae of Artemia fed with the chlorellae were used as an infecting vector. Of the 17 strains, seven formed stable symbioses and one formed a transient infection that disappeared within several weeks. Chlorellae of the nine other strains were cleared out of the infected hydra within 2-3 days. There was a distinct correlation between the ability of the chlorellae to form stable symbioses and their ability to adapt and grow in media enriched with 0.5% proteose peptone. Only strains that grew in the latter medium formed symbioses with the hydra. The symbioses formed with the different strains of chlorellae differed from one another. Hydra infected with some strains greened completely while those infected with other strains greened only partially. The degree of infection varied also within each population, and there were differences in the distribution of the various chlorellae along the stalk and inside the digestive cells of the hydra. Growth rates of the infected hydra were all less that those of aposymbiotic hydra or of hydra hosting native zoochlorellae. We conclude that adaptability to a nutrient-rich environment inside the perialgal vacuole of the digestive cell and a sufficient growth rate therein are crucial to the ability of chlorellae to form stable symbioses with H. viridis. In time, co-adaptation of hydra and chlorellae would restore the normal growth rate of the former and bring about regularity to the form and extent of infection by the latter.

Cells, Cultured

The cryopreservation of Chlorella. 1. Interactions of rate of cooling, protective additive and warming rate.

The cryoprotective additives glycerol and dimethylsulphoxide were found to be toxic to Chlorella cells at concentrations greater then 2.5% w/v. Polyvinylpyrrolidone, was not damaging up to a concentration of 15%w/v. Chlorella 211/7a had a recovery rate greater than 95% at all rates of cooling studied. With Chlorella 211/8h the survival was lower than 0.1% at all rates examined. The addition of dimethylsulphoxide (5% w/v) to Chlorella 211/8h increased the recovery, particularly at the faster rates of cooling; with polyvinylpyrrolidone (10% w/v) there was an optimum range of cooling rate. Cells of Chlorella 211/7a from the exponential phase of growth were found to be damaged both by a temperature reduction from 25 degrees C to 0 degrees C (thermal shock) and by freezing and thawing. In contrast cells from the stationary phase of growth were resistant to these stresses.

Cell Division

Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase.

A partial cDNA clone coding for the haem-binding domain of NADH:nitrate reductase (EC 1.6.6.1) (NR) from the unicellular green alga Chlorella vulgaris has been isolated, sequenced and expressed. A 1.2 kb cDNA (pCVNR1) was isolated from a lambda gt11 expression library produced from polyadenylated RNA extracted from nitrate-grown Chlorella cells. pCVNR1 hybridized to a 3.5 kb mRNA transcript that was nitrate-inducible and absent from ammonium-grown cells. The entire sequence of pCVNR1 was obtained and found to have a single uninterrupted reading frame. The derived amino acid sequence of 318 amino acids has a 45-50% similarity to higher-plant NRs, including Arabidopsis thaliana, spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum). A comparison with the putative domain structure of higher-plant nitrate reductases suggested that this sequence contains the complete haem-binding domain, approximately one-third of the Mo-pterin domain and no FAD-binding domain. A 32% sequence similarity is evident when comparing the Chlorella NR haem domain with that of calf cytochrome b5. Expression of pCVNR1 in a pET vector synthesized a 35 kDa protein that was antigenic to anti-(Chlorella NR) antibody. The spectral properties of this protein (reduced and oxidized) in the 400-600 nm region are identical with those of native Chlorella NR and indicate that haem is associated with the protein.

Amino Acid Sequence

Detection of Chlorella-specific IgE in mould-sensitized children.

The content of IgE, specific to the unicellular green alga Chlorella sp., was analysed in sera from 46 atopic children sensitized to moulds, using radioallergosorbent test (RAST), immunoblotting and crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis (CIE/CRIE). Chlorella-specific IgE was found in 23/46 sera by RAST, in 28/41 sera by immunoblotting and in 6/30 sera by CIE/CRIE. The Chlorella components most frequently binding IgE as analysed by gradient gel electrophoresis and immunoblotting were of molecular weights of approximately 13, 17, 19, 26 and 49 kD. Twenty-nine precipitating antigens, including seven IgE-binding precipitates were detected by CIE/CRIE. The study shows that low concentrations of specific IgE are formed to the green alga Chlorella in sera from atopic individuals sensitized to moulds.

Adolescent

The determination of the membrane ptoential of Chlorella vulgaris. Evidence for electrogenic sugar transport.

From data on the accumulation of tetraphenylphosphonium within Chlorella vulgaris cells, it can be estimated that these cells possess a membrane potential of --120 to --150 mV (inside negative). Under anaerobic conditions as well as in the presence of uncoupling agents the membrane potential drops to about -60 to -80 mV. Nystatin (50 mug/ml) abolishes it almost completely. Since it took more than 1 h before the tetraphenylphosphonium equilibrium was reached, this method could not be used to measure relatively fast transient changes in membrane potential. However, the rate of influx of tetraphenylphosphonium is also directly dependent on membrane potential and can be followed within minutes. Using this phenomenon as an indicator for membrane potential a brief transient depolarisation was detected after the addition of sugars taken up by Chlorella via the proton cotransport system. The depolarisation was absent from cells not induced for sugar uptake and induced cells did not show it with substances not transported, like mannitol. The maximal depolarisation observed amounted to about 70 mV; after 1 min, however, the membrane potential returned to a value about 25 mV less negative than the one before sugars was added. The results demonstrate that sugar uptake in Chlorella is electrogenic. The delta pH plus membrane potential measured for Chlorella completely cover the energy required to explain the 1600-fold accumulation of 6-deoxyglucose experimentally observed.

Anaerobiosis

Competition between Chlorellae in chimeric infections of Hydra viridis: the evolution of a stable symbiosis.

Aposymbiotic polyps of Hydra viridis were infected with one or two of the following strains of Chlorella: the native strain obtained from green H, viridis, and the originally non-symbiotic strains Fs and 211/8p cultured in vitro. Larvae of Artemia served as infecting vectors. Chimeric infections were obtained with two different Chlorella strains cohabiting in the same cells and polyps. In time, the chimeric infections disappeared and mixed populations of Hydra were formed with different strains of Chlorella in different polyps. We suggest that the Chlorella/Hydra symbiosis was initiated originally by an infection of preadapted hydra by preadapted chlorellae. Through intracellular interalgal competition and competition between dissimilar infected cells and polyps, the present-day stable symbiosis has evolved.

Animals

[Physico-chemical determination of the ploidy of the unicellular alga, Chlorella pyrenoidosa (strain 211/8b) (author's transl)].

The ploidy of the unicellular green alga Chlorella pyrenoidosa (strain 211/8b) has been determined by means of renaturation kinetics. The nuclear DNA is made up from fast, intermediate and slow renaturing sequences, which represent respectively about 5, 15 and 80% of the DNA. These observations are consistent with the findings in other eukaryotic nuclear DNAs. Nevertheless, the relative importance of the repeated sequences is much lower than that observed in Chlamydomonas reinhardi [16] and in higher plants [18-20], but slightly higher than that obtained in Chlorella vulgaris [17]. The kinetic complexity of the main fraction of the Cl. pyrenoidosa nuclear DNA is found to be 2.94 - 10-10 daltons (mean value of five independant experiments) assuming value of 2.1 - 10-8 daltons for Cl. pyrenoidosa chloroplastic DNA. When compared with the analytical complexity of this fraction (80% of the nuclear DNA analytical complexity, that is 2.02 - 10-10 daltons), one can assume that the slow renaturing fraction of the nuclear DNA is constituted by a unique nucleotide sequence. This result thus suggests that Cl. pyrenoidosa (strain 211/8b) is an haploid organism. The possible existence of an haploid genome in the nuclei of the algae from Chlorella genus and the apparent absence of sexuality might explain the high discrepancy observed in the G + C content of the Chlorella nuclear DNAs.

Chlorella