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Poly(A) RNA populations, polypeptide synthesis and macromolecule accumulation in the cell cycle of the eukaryote Chlorella.

Synchronous cultures of Chlorella, that were obtained with minimum metabolic perturbation by centrifugal selection, reveal that progress through the cell cycle requires no change in the poly(A)+ mRNA population, although changes do occur during nutritional adaptation. Of the abundant soluble proteins, 93% are synthesized continuously through the cell cycle and those that are discontinuous show similar patterns in control cells. The synthesis of proteins is compared with parallel studies of accumulation of enzyme activity and it is shown that there is no discrepancy in their pattern of accumulation when both are studied under the same culture conditions. The eukaryote cell cycle can allow stable relative rates of synthesis of most proteins and balanced rates of accumulation of most enzyme activities. Macromolecule classes differ in their rates of accumulation throughout the cell cycle: total RNA increases linearly, poly(A)+ RNA accumulation is restricted to G1 phase, but total protein accumulation accelerates smoothly through G1, S and mitosis phases, pausing at cytokinesis. There is no evidence that the cell cycle requires an extensive programme of differential enzyme synthesis. The cycle can therefore proceed with minimum disturbance of metabolism required for growth.

Cell Cycle↗

The complexity of poly(A)+ RNA in the alga Chlorella fusca.

RNA excess hybridization with radioactively labelled complementary DNA (cDNA) was used to reveal the complexity of poly(A)+ RNA from Chlorella fusca var. vacuolata. RNA was tested from cells during photosynthetic exponential growth and during adaptation to heterotrophic growth in the dark on acetate. Both RNA populations were resolved into abundant, intermediate and rare sequence classes. Abundant sequences (200-400 copies per cell) constituted a significantly larger proportion of total poly(A)+ RNA in acetate-adapting cells than in exponentially growing autotrophic cells. Both types of RNA contained a rare sequence class of complexity consistent with a composition of about 20 000 different sequences. This indicated a substantially greater complexity of genes expressed in this alga and Euglena than in fungi and slime moulds. Heterologous hybridization, and hybridization with fractionated cDNA, showed that the majority of differences between RNA populations from exponentially growing and adapting cells were changes in relative abundance of groups of sequences, rather than presence of different sets of sequences in the two populations.

Acetates↗

The cell cycle of symbiotic Chlorella. III. Numbers of algae in green hydra digestive cells are regulated at digestive cell division.

Regression analysis of the relationship between the size of interphase and mitotic digestive cells of green hydra, and the numbers and total volume of the symbiotic Chlorella algae they contain showed a partial correlation only, suggesting that numbers of algae per cell are not regulated by limiting them to a specific proportion of the host cell, and that the variation observed in numbers of algae per cell is not due to variation in host cell size. After hydra were fed, which stimulates algae and digestive cells to divide at the same time, numbers of algae per cell were higher in prophase than in interphase cells, and numbers increased as mitosis proceeded. In excised regenerating peduncles algae divide before digestive cells, and at the onset of digestive cell division mitotic cells were found to contain almost twice the number of algae as before excision. Thus, almost all of the algal cell division necessary to maintain a constant population size was associated with digestive cell division. Analysis of variance in numbers of algae in telophase mother and daughter cells suggested that the proportion of algae dividing as a result of host cell mitosis was greater in digestive cells with few algae than in those with many algae. The fact that the mechanism controlling the proportion of algae dividing in host cells is expressed at host cell division and is manifested in the daughter cells may contribute to wide variation in numbers of algae per cell.

Animals↗

Inhibitory effects of sterols isolated from Chlorella vulgaris on 12-0-tetradecanoylphorbol-13-acetate-induced inflammation and tumor promotion in mouse skin.

Inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice was observed in the methanol extract of Chlorella vulgaris, a green alga. The hexane soluble fraction obtained from the methanol extract exhibited marked inhibitory activity from which were isolated two delta(5,7)-sterols (ergosterol and 7-dehydroporiferasterol), two delta(5,7,9(11))-sterols [7,9(11)-dehydroergosterol and 7,9(11)-bisdehydroporiferasterol], two 5 alpha, 8 alpha-epidioxy-delta(6)-sterols (ergosterol peroxide and 7-dehydroporiferasterol peroxide), and a 7-oxo-delta(5)-sterol (7-oxocholesterol), among others. The delta 5'7-sterols, 5 alpha, 8 alpha-epidioxy-delta(6)-sterols and 7-oxo-delta 5-sterol inhibited TPA-induced inflammation in mice. The 50% inhibitory dose of these compounds for TPA-induced inflammation was 0.2-0.7 mg/ear. Furthermore, ergosterol peroxide markedly inhibited the tumor-promoting effect of TPA in 7,12-dimethylbenz[a]anthracene-initiated mice.

9,10-Dimethyl-1,2-benzanthracene↗

Biotransformation of aristolane- and 2,3-secoaromadendrane-type sesquiterpenoids having a 1,1-dimethylcyclopropane ring by Chlorella fusca var. vacuolata, mucor species, and Aspergillus niger.

Biotransformation of the aristolane-type sesquiterpene hydrocarbon (+)-1(10)-aristolene (1) from the crude drug Nardostachys chinensis and of the 2,3-secoaromadendrane-type sesquiterpene lactone plagiochilide (2) from the liverwort Plagiochila fruticosa by three microorganisms, Chlorella fusca var. vacuolata, Mucor species, and Aspergillus niger was investigated. C. fusca var. vacuolata and Mucor sp. introduced oxygen function into the cyclohexane ring of aristolene while A. niger oxidized stereoselectively one methyl of the 1,1-dimethyl group on the cyclopropane ring of aristolanes and 2,3-secoaromadendrane to give C-12 primary alcohol and C-12 carboxylic acid. The possible metabolic pathway of the formation of new metabolites is discussed. The stereostructures of new metabolites were established by a combination of NMR spectroscopy including HMBC and NOESY, X-ray crystallographic analysis, and chemical reaction.

Aspergillus↗

Purification and characterization of extracellular cysteine protease inhibitor, ECPI-2, from Chlorella sp.

An extracellular cysteine protease inhibitor (ECPI-2) was purified to homogeneity from the culture filtrate of Chlorella sp. 4533 by the combination of various column chromatographies. The molecular mass of the inhibitor was estimated to be 340 kDa by SDS-PAGE. The inhibitor was extremely heat-stable under acidic or neutral condition. ECPI-2 exhibited an inhibitory activity against the proteolytic activity of papain, ficin, or chymopapain, but not against stem bromelain or cathepsin B. The inhibitor showed no inhibitory activity against trypsin, alpha-chymotrypsin or thermolysin. ECPI-2 contains 33.6% carbohydrate residues by weight and inhibits papain at a molar ratio of 1:2. The proteolysis of the inhibitor by trypsin or alpha-chymotrypsin was apparent, but the inhibitory activity of ECPI-2 was unaffected by these enzymes. The alpha-chymotrypsin hydrolysis product from ECPI-2 was further separated into six fractions by gel filtration. From these results, it is suggested that ECPI-2 has several reactive sites for papain.

Algal Proteins↗

Evaluation of radioprotective action of a mutant (E-25) form of Chlorella vulgaris in mice.

The possible role of orally fed Chlorella vulgaris (E-25) in modulating the gamma-ray induced chromosomal damage in whole-body irradiated mice was evaluated using a micronucleus test. Different doses of E-25 were administered either chronically (once, twice or thrice a day for 28 days) or as single acute doses before/after irradiation. A significant radioprotective effect was observed in both acute and chronic pretreatments, but only at doses above 400 mg/kg body weight. However, in mice that received E-25 (500 mg/kg) three times a day for 28 days, there was no protective effect, and a significant loss in their body weight was observed. Interestingly, E-25 afforded significant radioprotection even when it was administered within 0.4 hr after irradiation.

Animals↗

Thymine dimer-dependent incision on ultraviolet light damaged-DNA in cell-free extracts of Chlorella pyrenoidosa.

Cell-free extracts of the unicellular algae Chlorella pyrenoidosa induce a UV-dependent incision of both phi x 174 RFI DNA and a 27-mer double-stranded oligonucleotide containing a single thymine dimer, as analyzed by agarose and denaturing polyacrylamide gel electrophoresis, respectively. The incision reaction occurred only on the thymine dimer-containing strand of the 27-mer, and a major incision product of 17-mer was observed. Addition of ATP or ATP-regenerating system to algal extracts increased the effect of DNA incision.

Base Sequence↗

Stereocontrolled reduction of alpha- and beta-keto esters with micro green algae, Chlorella strains.

The stereocontrolled reduction of alpha- and beta-keto esters using micro green algae was accomplished by a combination of the cultivation method and the introduction of an additive. The reduction of ethyl pyruvate and ethyl benzoylformate by the photoautotrophically cultivated Chlorella sorokiniana gave the corresponding alcohol in high e.e. (>99% e.e. (S) and >99% e.e. (R), respectively). In the presence of glucose as an additive, the reduction of ethyl 3-methyl-2-oxobutanoate by the heterotrophically cultivated C. sorokiniana afforded the corresponding (R)-alcohol. On the other hand, the reduction in the presence of ethyl propionate gave the (S)-alcohol. Ethyl 2-methyl-3-oxobutanoate was reduced in the presence of glycerol by the photoautotrophically cultivated C. sorokiniana or the heterotrophically cultivated C. sorokiniana to the corresponding syn-(2R,3S)-hydroxy ester with high diastereo- and enantiomeric excess (e.e.). Some additives altered the stereochemical course in the reduction of alpha- and beta-keto esters.

Chlorella↗

Characterization and amino acid sequences of cytochromes c6 from two strains of the green alga Chlorella vulgaris.

Cytochromes c6 from the green algae Chlorella vulgaris CK-5 (CK5cyc6) and C. vulgaris CK-22 (CK22cyc6) were characterized and their amino acid sequences were analyzed. CK5cyc6 had a molecular mass of 9.3 kDa, isoelectric points of 3.0 (reduced) and 3.6 (oxidized), and a redox potential of +362 mV at pH 7.0. CK22cyc6 had a molecular mass of 9.5 kDa, isoelectric points of 2.9 (reduced) and 3.5 (oxidized), and a redox potential of +355 mV at pH 7.0. The absorption spectra of both cytochromes c6 showed 4 maxima in reduced form, and 2 maxima and a weak peak at 695 nm in oxidized form. The pyridine ferrohemochrome spectra indicated that their prosthetic group was heme c. These physicochemical properties were similar to those of other algal cytochromes c6. The amino acids (88 residues) of CK5cyc6 and CK22cyc6 were sequenced and the sequence motif -CXXCH-, which is typical of the heme-binding site of c-type cytochrome, was clearly confirmed in both cytochromes. Twenty-six amino acid residues were substituted, and the similarity score of each of them was 70.45%.

Algal Proteins↗

Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27.

Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.

Amino Acid Sequence↗

Chlorophyll derived from Chlorella inhibits dioxin absorption from the gastrointestinal tract and accelerates dioxin excretion in rats.

We investigated the effects of chlorophyll derived from Chlorella on gastrointestinal absorption of seven types of polychlorinated dibenzo-p-dioxin (PCDD) and 10 types of polychlorinated dibenzofuran (PCDF) in Wistar rats. Twenty-eight rats were randomly distributed into seven groups (n = 4). After overnight food deprivation, rats were given 4 g of the basal diet or 4 g of the chlorophyll diet containing 0.01-0.5% chlorophyll one time on day 1; each diet also contained 0.2 mL PCDD and PCDF standard solutions. The amounts of fecal excretion of PCDD and PCDF congeners from days 1 to 5 in the group fed 0.01% chlorophyll were 64.8% for 1,2,3,7,8-pentaCDD, 78.6% for 1,2,3,4,7,8-hexaCDD, 73.5% for 1,2,3,6,7,8-hexaCDD, 58.5% for 1,2,3,7,8,9-hexaCDD, 33.3% for 1,2,3,4,6,7,8-heptaCDD, 85.7% for 1,2,3,7,8-pentaCDF, 77.3% for 2,3,4,7,8-pentaCDF, 88.6% for 1,2,3,4,7,8-hexaCDF, 78.0% for 1,2,3,6,7,8-hexaCDF, 62.5% for 1,2,3,7,8,9-hexaCDF, 84.1% for 2,3,4,6,7,8-hexaCDF, 41.7% for 1,2,3,4,6,7,8-heptaCDF, and 40.0% for 1,2,3,4,6,7,8-heptaCDF greater (p < 0.01) than those of the control group, respectively. The fecal excretion of PCDD and PCDF congeners was remarkably increased along with the increasing dietary chlorophyll. The amounts of PCDD and PCDF congeners in rats on day 5 administered dioxin mixtures were lower in the 0.01% chlorophyll group than in the control group, ranging from 3.5 to 50.0% for PCDD congeners and from 3.7 to 41.7% lower for PCDF congeners, except for 2,3,7,8-tetrachlorodibenzofuran. The amount of PCDD and PCDF congeners in rats was remarkably decreased along with the increasing dietary chlorophyll. These findings suggest that chlorophyll is effective for preventing dioxin absorption via foods.

Animals↗

[Expression of rabbit neutrophile peptide-1 in nitrate reductase-deficient mutant of Chlorella ellipsoidea].

Application of transgenic Cholrella as bioreactor to express rabbit neutrophile pepetide-1 (NP-1) shows great practical value. In this paper, an NP-1 expression vector containing two selective marker genes NPTII and nitrate reductase gene was constructed. The NP-1 gene was transformed into the nitrate reductase-deficient mutant nrm-4 of Chlorella ellipsoidea via electroporation, and the transgenic alga expressed the active NP-1 were obtained.

Animals↗

Methods to enhance tolerances of Chlorella KR-1 to toxic compounds in flue gas.

Possible methods to minimize the toxic effects of SOx and NOx on the growth of a highly CO2 tolerant and fast-growing microalga, Chlorella sp. KR-1, were investigated. Maintaining the pH of the culturing media at an adequate value was quite important to enhancing the tolerances of the microalgae to SOx and NOx. Controlling the pH by adding an alkaline solution, using a low flow rate of gas fed to the culture, and using a high concentration of inoculating cells were effective methods to maintaining the proper pH of the culture. Controlling the pH was the most effective method but could be applied only for some specific microalgae.

Air Pollutants↗

High photosynthetic productivity of green microalga Chlorella sorokiniana.

The batch culture of a newly isolated strain of a green microalga, Chlorella sorokiniana, was carried out using a conical helical tubular photobioreactor. The isolate was capable of good growth at 40 degrees C under an airstream enriched with 10% CO2. The maximum photosynthetic productivity was 34.4 g of dry biomass/(m2 of installation area x d) (12-h light/12-h dark cycle) when the cells were illuminated with an average photosynthetic photon flux density (photosynthetically active radiation ([PAR] 400-700 nm) simulating the outdoors in central Japan (0.980 mmol photons/[m2 x s]). This corresponded to a photosynthetic efficiency of 8.67% (PAR), which was defined as the percentage of the light energy recovered as biomass (394 kJ/[reactor x d]) to the total light energy received (4545 kJ/[reactor x d]). A similarly high photosynthetic efficiency (8.12% [PAR]) was also attained in the combined presence of 10% CO2, 100 ppm of NO, and 25 ppm of SO2. Moreover, good photosynthetic productivity was also obtained under high temperature and high light intensity conditions (maximum temperature, 46.5 degrees C; 1.737 mmol photons/[m2 x s]), when simulating the strong irradiance of the midday summer sun. This strain thus appears well suited for practical application for converting CO2 present in the stack gases emitted by thermal power plants and should be feasible even during the hot summer weather.

Base Sequence↗

Temperature dependence of the O2-oscillation pattern in the filamentous cyanobacterium Oscillatoria chalybea and in Chlorella kessleri.

Five characteristic discontinuities of the pattern of oxygen evolution have been detected for the filamentous cyanobacterium Oscillatoria chalybea in the temperature range of 0 degree C to 30 degrees C. The temperatures at which these discontinuities occur are: approximately 5 degrees C, approximately 11 degrees C, approximately 15 degrees C, approximately 21 degrees C and approximately 25 degrees C. The calculated initial 5-S state distribution, the miss parameter and the fraction of the fast transition S3-->So + O2 are affected. The discontinuities are observed at the same transition temperature also for Chlorella kessleri hence are not specific for the cyanobacterium. Based on these studies it is concluded that the not vanishing oxygen signal under the first flash of a flash train in Oscillatoria cannot have its origin in interactions between oxygen-evolving complexes. A decrease of temperature should slow down the expected charge exchanges, improve the oscillations thus reduce or lower the first two oxygen amplitudes of the oscillatoria pattern. Lowering of the temperature improves the oscillations but does not lower the first O2 signal of the pattern.

Activity Cycles↗

Effects of chlorella alkali extract on blood pressure in SHR.

When chlorella was treated with alkali after decolorization by methyl alcohol and then fractionated by gel filtration, the fraction showed a depressive effect. The blood pressure of SHRSP and SHR showed a fall of an average of 63 mmHG 1 hour after intravenous administration, 47 MMHg 2 hours after intraperitoneal administration. The blood pressure of normotensive Wistar rats also showed a fall of an average of 32 mmHg 1 hour after intravenous administration.

Alkalies↗

Association mechanisms of Europium(III) and Curium(III) with Chlorella vulgaris.

The association of Europium(III) (Eu[III]) and Curium(III) (Cm[III]) with Chlorella vulgaris and with cellulose was studied by a batch method and time-resolved laser-induced fluorescence spectroscopy (TRLFS). The kinetics study performed by the batch method showed that maximum adsorption of Eu(III) and Cm(III) on C. vulgaris was attained within 3 min of contact; afterward, the percentage adsorption decreased with time due to chelation of the ions with exudates released from C. vulgaris with a strong affinity for Eu(III) and Cm(III). The TRLFS revealed that the short-term adsorption of Eu(III) on C. vulgaris was attributable to its coordination with cellulose on the algal cell wall. However, Eu(III) coordinated with the functional groups of cellulose very weakly despite the large distribution coefficients observed. These results indicate that the reactions, both at the cell's surfaces through adsorption and in solution phases through chelation with the exudates, are important in estimating the behavior of Eu(III) and Cm(III) in aqueous environments.

Adsorption↗