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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↗

RNA triphosphatase component of the mRNA capping apparatus of Paramecium bursaria Chlorella virus 1.

Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5' diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the gamma-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.

Acid Anhydride Hydrolases↗

Potassium ion channels of Chlorella viruses cause rapid depolarization of host cells during infection.

Previous studies have established that chlorella viruses encode K(+) channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs(+) to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t(1/2)], about 9 min) and slowest with virus NY-2A (t(1/2), about 12 min). Cs(+) inhibited membrane depolarization only in viruses that encode a Cs(+)-sensitive K(+) channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K(+) conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.

Cell Membrane↗

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↗

Effect of docosahexaenoic acid-fortified Chlorella vulgaris strain CK22 on the radial maze performance in aged mice.

Effect of docosahexaenoic acid (DHA) [22: 6(n-3)]-fortified Chlorella oil fraction on radial maze performance was studied in aged mice. Male ICR mice aged 9 months were fed a diet containing 2 g DHA-fortified Chlorella oil fraction/100 g diet or normal diet (Control group) for 2 months. Two months after the start of feeding, the mice were tested for learning ability related to 2 types of memory, reference memory and working memory, with the partially (4 of 8) baited eight-arm radial maze. Reference memory is a kind of information that should be retained until the next trial. Working memory is a kind of information that disappears in a short time. Entry into the unbaited arms and repeated entry into the visited arms were defined as reference memory errors and working memory errors, respectively. DHA-fortified Chlorella oil fraction administration to mice for 2 months resulted in a significant decrease in the number of working memory errors without affecting the number of reference memory errors. A significant increase in the DHA content in the brain was also observed. These results suggest that the intake of DHA-fortified Chlorella oil fraction effectively enhances working memory in maze performance.

Aging↗

Uptake and accumulation of exogenous docosahexaenoic acid by Chlorella.

Tuna oil or its hydrolysate was added to a culture of Chlorella for its nutritional fortification as a feed for rotifer. Exogenous docosahexaenoic acid (DHA) in its free form was taken up by the cells of Chlorella vulgaris strain K-22 and by other strains, but tuna oil was not taken up by the cells. Accumulated DHA was found by electron microscopy in the cells in oil droplets. All strains of Chlorella used in these experiments took up exogenous DHA into the cells. It seems that the structure of the cell wall did not affect the uptake of DHA into the Chlorella cells.

Animals↗

[The regulation activity of Chlorella virus gene 5' upstream sequence in Escherichia coli and eucaryotic alage].

The 5' upstream regions of adenine methyltransgerase gene and major coat protein gene (PAMT, PVP54) in Chlorella virus genomes were used to contract transformation vectors in E. coli and eukaryotic algae. The regulation activities of PAMT and PVP54 comparing with PRPL and CaMV35S promoters were analyzed in different E. coli strains and Chlorella species. It is found that the luciferase activity controlled by PAMT is 50-400 times higher than that controlled by PRPL. The regulation activity of PAMT in 2 Chlorella species is obviously higher than that of CaMV35S promoter. It is the first report that the 5' upstream region of Chlorella virus gene has strong regulation activity in eucaryotic algae. The result suggests this regulation sequence will have an excellent application in the eucaryotic algae genetic engineering.

Chlorella↗

[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↗

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana.

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

Biological Transport↗

Effect of different densities of live and dead Chlorella vulgaris on the population growth of rotifers Brachionus calyciflorus and Brachionus patulus (Rotifera).

In order to maintain rotifer populations during periods of low algal production, it is necessary to offer alternate diets, some of which include forms of preserved algae. The present work is based on the effect of live and dead Chlorella vulgaris on the population growth of Brachionus calyciflorus and Brachionus patulus. The experimental design consisted of three algal levels (0.5 x 10(6), 1.5 x 10(6) and 4.5 x 10(6) cells ml-1) offered in three forms (living, frozen and heat-killed). The maximal population density values for B. calyciflorus ranged from 55 +/- 1 ind. ml-1 (at 0.5 x 10(6) cells ml-1) to 471 +/- 72 ind. ml-1 (at 4.5 x 10(6) cells ml-1) with live Chlorella, but was much lower (6 +/- 1 to 26 +/- 6 ind. ml-1) with frozen or heat-killed alga under comparable food levels. However, the maximum population density of B. patulus under live or or heat-killed Chlorella was similar at comparable algal levels but when offered frozen algae it was four times less. The highest mean peak population density was 1,277 +/- 83 ind. ml-1 under 4.5 x 10(6) cells ml-1. The rate of population increase for B. calyciflorus varied from 0.50 to 0.79 using live Chlorella, but under comparable conditions, this range was lower (0.21 to 0.31) for B. patulus. Results have been discussed in light of possible application for aquaculture.

Animal Feed↗

Toxicity of metal mixtures to a tropical freshwater alga (Chlorella sp): the effect of interactions between copper, cadmium, and zinc on metal cell binding and uptake.

The individual and combined effects of copper, cadmium, and zinc on the cell division rate of the tropical freshwater alga Chlorella sp. were determined over 48 to 72 h. Metal mixtures were prepared based on multiples of their single-metal median effective concentration (EC50) values, i.e., toxic units (TU) using a triangular mixture design with five toxicant levels (0, 0.75, 1.0, 1.25, and 1.5 TU). Single-metal EC50 values after a 72-h exposure were 0.11, 0.85, and 1.4 microM for copper, cadmium, and zinc, respectively. Significant interactions were observed for all metal combinations after 48 and 72 h. An equitoxic mixture of Cu + Cd was more than concentration additive (synergistic) to the growth of Chlorella sp., while combinations of Cu + Zn, Cd + Zn, and Cu + Cd + Zn were all less than concentration additive or were antagonistic. To determine the effect of each metal on the uptake of the other, extracellular (membrane-bound) and intracellular metal concentrations, both alone and in mixtures, were compared. The increased growth inhibition observed for mixtures of Cu + Cd was due to higher concentrations of cell-bound and intracellular copper in the presence of cadmium compared with copper alone (i.e., cadmium-enhanced copper uptake). In contrast, both extra- and intracellular cadmium concentrations were reduced in the presence of copper. In mixtures of Cu + Zn, copper also inhibited the binding and cellular uptake of zinc, which resulted in decreased toxicity. Zinc had no appreciable effect on the uptake of copper by Chlorella sp. Our results suggest that all three metals share some common uptake and transport sites on Chlorella cells and that copper out competes both cadmium and zinc for cell binding. Determination of metal cell distribution coefficients (K(d)) confirmed that K(d) values for cadmium and zinc in single-metal exposures decreased in the presence of copper.

Cadmium↗

Isolation and characterization of Chlorella viruses from freshwater sources in Korea.

We isolated 23 Chlorella viruses from 9 Korean cities. The viruses were initially amplified in the Chlorella strain NC64A. Pure isolates were obtained by repeated plaque isolations. A SDS-PAGE analysis revealed similar but distinct protein patterns, both among the group of purified viruses and in comparison with the prototype Chlorella virus PBCV-1. Digestions of the 330- to 350-kb genomic DNAs with 10 restriction enzymes revealed different restriction fragment patterns among the isolates. One isolate, SS-1, was resistant to digestion with HindIII, PvuII, AluI, and HaeIII, indicating methylation at the AGCT or GC sequences. Some isolates reacted with antiserum against PBCV-1. The others that did not react to this PBCV-1 antibody reacted to the antibody that was raised against purified HS-2 virion. The tRNA-coding regions of 8 Chlorella viruses were cloned and sequenced. These viruses contained 14-16 tRNA genes within a 1.2- to 2-kb region, except for the SS-1 isolate, which had a 1039-bp spacer in a cluster of 11 tRNA genes. The SS-1 spacer contained an open-reading frame (ORF) of 294 amino acids. This ORF had a 51% amino acid sequence similarity to the PBCV-1 ORF A478L. A Southern blot analysis suggested that it was a novel gene that lacked a homologue in PBCV-1.

Amino Acid Sequence↗

[Immunogold localization of ribulose-1,5-bisphosphate carborylsae/oxygenase in chloroplasts of Chlorella].

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a first key enzyme in the Calvin Circle of plant cell photosynthesis. This paper mainly studied gold immunolocalization of Rubisco of Chlorella spp. 640909, and the Native-PAGE and, SDS-PAGE and Western bloting analysis, as well as the observation to pyrenoid ultra structure. The Native-PAGE result showed a main band, evidenced as the Rubisco band by the Western blot with the antibody against the Rubisco from C. prototothecoides, The special immunoacton of Rubisco from Chlorella spp. 640909 and the antibody to large subunit of Rubisco from C. prothecoides showed the large subunit proteins of Rubisco in the two species of Chlorella shared the high homology. The SDS-PAGE and Western blotting maps showed the molecule weight of the large subunit of Rubisco of Chlorella spp. 640909 was about 55 KD. The shape of pyrenoid ultra structure of the electronic microscope was oblong, and was embedded in starch sheath, with 2 swelling thylakoids through out a center portrait channel of the pyrenoid. There were some connections between pyrenoid and the chloroplast stroma. The distribution of the large subunits and the whole Rubisco in the chloroplast of Chrolella spp. 640909 was studied by immunoelectron microscopy by embedded sections with antibody to large subunit and whole enzyme followed by second antibody, goad anti-rabbit immunoglobulin G conjugated to 10 nm gold particles(Sigma production). The result showed the antibodies against large subunit and whole enzyme heavily labeled the pyrenoid, as well as starch sheath region, whereas the thylakoid region of the plastid was lightly labeled. And the whole Rubisco antibody labeled the pyrenoid surface more heavily than the large subunit antibody did. It is demonstrated the pyrenoid and starch sheath have the photosynthesis function. Rubisco concentrating in pyrenoid and starch sheath is valuable to fix CO2 for photosynthesis in algae.

Blotting, Western↗

[Chinese Chlorella viruses and their molecular biological properties].

An extensive survey for Chlorella viruses revealed that Chlorella viruses are widely distributed over China. Eleven isolates of Chlorella viruses have been isolated, which lysis the Chlorella viruses sp. (strain NC64A). These isolates were named BJ-1, BJ-2, BJ-3, BJ-4, FJ-1, FJ-2, NJ-1, CDT-1, HCJ-1, SCB-1 and SCC-1 respectively. They have several common properties, including polyhedral morphology, and linear dsDNA genomes. However, the restriction patterns of viral DNA, the concentration of m5dC and m6dA in viral DNA, and the composition of viral structure protein are different among these isolates. All of viral major capsid proteins are 54,000 except FJ-1, whose major capsid protein is smaller than 54,000. Western blot analysis showed strong immunological cross reaction among all of viral proteins with PBCV-1's antiserum except FJ-1. It indicates that the homology between these isolates and PBCV-1 is high. Among these isolates, FJ-1 has some special properties.

Base Composition↗

Safety and immunoenhancing effect of a Chlorella-derived dietary supplement in healthy adults undergoing influenza vaccination: randomized, double-blind, placebo-controlled trial.

BACKGROUND: Enhancement of immune function has been claimed as a benefit of some natural health products, although few have been subjected to randomized clinical trials. We evaluated the effect of an oral dietary supplement derived from the edible microalga Chlorella pyrenoidosa on immune response after influenza vaccination. METHODS: We conducted a randomized, double-blind, placebo-controlled community-based clinical trial in a convenience sample of 124 healthy adults at least 50 years of age randomly assigned to receive the study product (200 or 400 mg of a Chlorella-derived dietary supplement) or placebo. Participants took the study product or placebo once daily for 28 days. On day 21, we administered a single dose of a licensed trivalent, inactivated influenza vaccine. We obtained serum specimens to measure hemagglutination inhibition titres before and 7 and 21 days after vaccination. The primary immunological outcomes were the proportion of participants with a 4-fold or greater increase in antibodies and geometric mean antibody titres after vaccination; the proportion of participants reporting adverse events during therapy was the safety outcome. RESULTS: A total of 117 (94%) participants completed all aspects of the study. There were no differences in the proportions of recipients of 200 or 400 mg of the Chlorella-derived dietary supplement or placebo who achieved at least a 4-fold increase in antibodies (proportions for the 3 virus strains ranged from 17.9% to 28.2% for the 200-mg group, from 11.1% to 22.2% for the 400-mg group and from 19.0% to 21.4% for the placebo group; p > 0.05 for all comparisons). Reports of adverse events were similar for recipients of the supplement and placebo, except with regard to fatigue, which was reported more frequently by recipients of 200 mg of the supplement (18/41 or 44%) than by those who received 400 mg of the supplement (8/40 or 20%; p = 0.032) or placebo (8/42 or 19%; p = 0.019). Recipients of 400 mg of the supplement who were 55 years of age or younger had significantly higher geometric mean antibody titres against influenza A/New Caledonia 21 days after vaccination (p = 0.047) and against B/Yamanashi 7 days after vaccination (p = 0.034); the trends were nonsignificant for titres against A/Panama. We also observed similar increases for the proportions of subjects with a 2-fold or greater or a 4-fold or greater increase in antibodies. INTERPRETATION: The Chlorella-derived dietary supplement did not have any effect in increasing the antibody response to influenza vaccine in the overall study population, although there was an increase in antibody response among participants aged 50-55 years. Adverse events were similar among those receiving the supplement and the placebo. Further studies are warranted to explore the range of clinical effects resulting from ingestion of this dietary supplement.

Adjuvants, Immunologic↗

[Progress on Chlorella in the area of molecular biology].

Unicellular green alga Chlorella is a kind of important eukaryotic microalga. A lot of work about Chlorella was reported in recent years with the rapid development of algal biotechnique. In the area of molecular biology, studies of Chlorella focus on the nuclear and chloroplast genome, cloning and analysis of important genes and genetic engineering using Chlorella as vector. This review reports the research progress in these aspects.

Animals↗

Regulation of ethylene-forming system in Chlorella by near-UV radiation.

The growth (cell division) of Chlorella cells cultured under photosynthetically active radiation (PAR) without near-UV radiation was inferior to that under PAR with near-UV radiation. To elucidate this phenomenon, the relationship between near-UV radiation and ethylene production in Chlorella cells was examined. The suppression of ethylene production by UV radiation suggests that this phenomenon is associated with the production of ethylene. Chlorella (a eukaryotic protista) was found to produce ethylene from methionine via S-adenosyl-methionine (SAM) and 1-aminocyclo-propane-1-carboxylate (ACC) as in higher plants, and the activity of ACC oxidase, a limiting factor of ethylene production, appears to be associated with the growth of Chlorella. A possible mechanism for the action of near-UV radiation on ethylene biosynthesis and growth suppression by ethylene is discussed.

Amino Acid Oxidoreductases↗