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Importance of the first external loop for substrate recognition as revealed by chimeric Chlorella monosaccharide/H+ symporters.

It had been shown previously by heterologous expression in Schizosaccharomyces pombe, that the two monosaccharide/H+ symporters HUP1 and HUP2 of Chlorella kessleri differ significantly concerning their substrate specificity: HUP1 transports predominantly D-glucose while HUP2 prefers D-galactose. Several chimeric transporters were constructed and their substrate specificities determined. Surprisingly, it is sufficient to replace the first part of the external loop 1 of the HUP1 symporter by the corresponding portion of HUP2 to improve transport and also to decrease the Km value for D-galactose. Additional data indicating the importance of the first loop for substrate recognition and binding are discussed.

Amino Acid Sequence↗

Anti-tumour-promoting glyceroglycolipids from the green alga, Chlorella vulgaris.

Two new monogalactosyl diacylglycerols were isolated from the freshwater green alga, Chlorella vulgaris, as anti-tumour promoters, together with three monogalactosyl diacylglycerols and two digalactosyl diacylglycerols. The new monogalactosyl diacylglycerol containing (7Z,10Z)-hexadecadienoic acid showed a more potent inhibitory effect toward tumour promotion than the other glycerolipids isolated.

Anticarcinogenic Agents↗

Structural proteins and lipids in a virus, PBCV-1, which replicates in a Chlorella-like alga.

PBCV-1, a large dsDNA-containing virus which replicates in a Chlorella-like green alga, is composed of approximately 64% protein, 25% DNA, and 5-10% lipid on a weight basis. Polyacrylamide gel electrophoresis of the dissociated virus particle resolves 50 to 60 proteins which range in apparent molecular weight from 10,000 to 135,000. Two of these proteins are glycoproteins and at least four are located on the viral surface. The major lipids are phosphatidyl choline, phosphatidyl ethanolamine, and an unidentified component. The effect of organic solvents, surfactants, and chelating and reducing agents on viral infectivity and ultrastructure are reported. Inhibitor studies established that PBCV-1 protein synthesis occurs on cytoplasmic ribosomes.

Chlorella↗

Infection of a Chlorella-like alga with the virus, PBCV-1: ultrastructural studies.

Ultrastructural studies revealed that the virus, PBCV-1, adsorbs to the surface of the Chlorella-like green alga NC64A and enzymatically digests a portion of the host cell wall. The viral DNA is then released into the interior of the cell leaving an empty capsid on the surface. Thus uncoating of the viral genome occurs at the surface of its host. PBCV-1 also adsorbs to and digests the host wall of either heat-killed, methanol-extracted, or purified cell wall fragments.

Chlorella↗

Lytic viruses infecting a Chlorella-like alga.

A number of viruses which form plaques on the unicellular, eukaryotic, Chlorella-like green alga, strain NC64A, were isolated from fresh water ponds and rivers in Illinois, North Carolina, and South Carolina. The viruses were large polyhedrons (160 to 190 nm in diameter) and contained dsDNA genomes of ca. 300 kbp. All of the viral DNAs hybridized with DNA from the previously described PBCV-1 virus. However, the viruses, even many of those isolated from the same water sample, could be distinguished from one another by DNA restriction endonuclease digestion. The viruses, including PBCV-1, were grouped into five classes by their resistance to certain DNA restriction endonucleases. Presumably the DNAs in the five classes contain different types or amounts of modified bases.

Chlorella↗

Infection of a Chlorella-like alga with the virus PBCV-1: transcriptional studies.

Infection of the unicellular, eukaryotic Chlorella-like green alga NC64A by the large dsDNA containing virus PBCV-1 immediately reduced host RNA synthesis. Chloroplast rRNAs, but not cytosolic rRNAs, were degraded following viral infection. Northern blot analysis utilizing four cloned fragments of PBCV-1 DNA as probes, which represent about 12% of the viral genome, revealed several properties of PBCV-1 transcription: A few viral transcripts were detected within 5 min after infection. Each PBCV-1 DNA clone hybridized to both early and late transcripts which implies that early and late genes are dispersed throughout the viral genome. The transition from early to late transcription occurred between 40 and 60 min after infection coincident with the onset of viral DNA synthesis. Three of the four DNA clones hybridized to transcripts which additively were larger than the corresponding DNA probe. This could reflect RNA processing, presence of overlapping genes, or transcription from both DNA strands. A few, but not all, early transcripts were synthesized in the presence of cycloheximide. This suggests that the virus either carries in its own RNA polymerase or uses a host RNA polymerase for very early viral transcription and that synthesis of additional, later transcripts depends on translation of an early gene product(s).

Chlorella↗

Properties of the Chlorella receptor for the virus PBCV-1.

The virus PBCV-1 attached rapidly, specifically, and irreversibly to the external surface of cell walls of its host, a unicellular, eukaryotic Chlorella-like green alga. Attachment was pH and salt dependent. Each cell contained at least 5 X 10(4) PBCV-1 binding sites and Scatchard analysis indicated that each cell could adsorb 5000 PBCV-1 particles. The PBCV-1 receptor was unaffected by extraction with organic solvents, detergents, high salts, or treatment with several proteases as well as the polysaccharide degrading enzymes, cellulase and pectinase. In contrast, acid and alkali treatments of walls at high temperatures and treatment with an enzyme preparation from PBCV-1 lysates destroyed the virus receptor. We suspect that the receptor is a carbohydrate.

Carbohydrates↗

Chlorella viruses contain linear nonpermuted double-stranded DNA genomes with covalently closed hairpin ends.

Pulsed field electrophoresis established that Chlorella viruses contain linear, nonpermuted, 330- to 380-kb dsDNA genomes. Terminal DNA restriction fragments of one virus, PBCV-1, were identified by Bal31 exonuclease digestion; the termini probably contain covalently closed hairpin ends. The end fragments cross-hybridize indicating terminal repetition; the region of repetition extends no more than 2.5 kb from the ends.

Chlorella↗

Effects of various organic chemicals on the regreening of red colored Chlorella zofingiensis.

Under nitrogen-deficient culture conditions Chlorella zofingiensis decomposes the chlorophylls, synthesizes secondary cartenoids, and thus develops red colored resting stages (akinetes). On transfer of the akinetes into nitrogen-containing fresh culture medium the algae regreen completely within a short period. The results of the experiments presented in this study show that the regreening process reacts with high sensitivity to the addition of environmentally hazardous chemicals. This system proved to be much more sensitive than other methods of testing for ecotoxicity. This screening test is universally applicable and offers, in addition, the possibility of testing volatile compounds since it is carried out in air-tight culture vessels. A phytotoxicity of 50% calculated as inhibition of the chlorophyll biosynthesis during exposure for 48 hr was found for paraquat at concentrations of 0.15 microM (added as methysulfate) and 0.28 microM (added as chloride) and for pentachlorophenol (PCP) at 0.16 microM. The chemicals 2- and 4-nitrophenol, however, proved to be much less toxic. Here, a 50% inhibition of chlorophyll biosynthesis was observed at 49 and 75 microM. When exposed to 4-chloroaniline even concentrations of 200 microM were not sufficient to cause a 50% inhibition of the regreening process. In additional experiments the influence of increasing salt concentrations (NaCl stress) and simultaneous exposure to the toxicants was examined. A step-by-step increase in the salinity of the experimental medium resulted in a drastic reduction of the toxicity caused by the addition of paraquat. This effect was much less pronounced when PCP was employed as toxicant. A different result was obtained with 2-nitrophenol: Here the toxic action was slightly enhanced. Differences in the time-dependent toxic response were also observed using the diverse toxicants. Probable causes are discussed.

Aniline Compounds↗

Influence of glucose amendment on the toxicity of two nitrophenols to Chlorella vulgaris.

Glucose amendment (0.5%) to the culture medium of Chlorella vulgaris reversed the algal toxicity caused by p-nitrophenol (PNP) and m-nitrophenol (MNP), only at their algistatic levels. Algistatic and algicidal concentrations of PNP and MNP, in the absence of glucose, led to the liberation of more nitrite in the culture medium. However, addition of glucose inhibited nitrite formation.

Cell Count↗

Effect of atrazine herbicide on growth, photosynthesis, protein synthesis, and fatty acid composition in the unicellular green alga Chlorella kessleri.

Sublethal atrazine concentrations induced a general inhibition on growth, photosynthesis, and dark respiration in the green alga Chlorella kessleri. 14C-protein hydrolysate indicated a maximum incorporation level by 5 microM atrazine after 48 hr. Progressive reduction in protein synthesis was associated with increasing herbicide concentration at all experimental periods. The herbicide had preferential effect on the associated fatty acid composition of total and phospholipids. After 24 and 48 hr the herbicide stimulated fatty acids synthesis at concentrations where photosynthesis was inhibited. Meanwhile, stearic and miristic acids disappeared at 15 microM after 24 hr and the total polyunsaturated fatty acids were not affected after 48 hr. Fatty acid synthesis was sensitive to treatment at 72 hr by 5 and 10 microM atrazine, whereas the total saturated fatty acids were completely inhibited.

Atrazine↗

Effect of reduced pyridine nucleotides and tungstate on the in vitro insertion of molybdenum into demolybdo-nitrate reductase of Chlorella vulgaris.

Demolybdo-nitrate reductase (cytochrome c reductase) (NADH: acceptor oxidoreductase, EC 1.6.99.3) of Chlorella vulgaris can be activated in vitro to nitrate reductase by insertion of Mo from molybdate into the apoprotein. Evidence is here presented that reduction of the enzyme by reduced pyridine nucleotides inhibits the process of molybdenum insertion. This report also describes the effect of molybdate and tungstate concentration on the activation process. The activation is sigmoidally related to molybdate concentration with a calculated Hill coefficient of NH = 3. At suboptimal molybdate concentrations, tungstate stimulates enzyme activation by molybdate; but at saturating molybdate concentrations, tungstate is inhibitory. These facts are regarded as an indication that molybdate and tungstate are both positive effectors of molybdenum incorporation, but that they are competitors for the active Mo center.

Apoproteins↗

Characterization of a protein kinase gene from two Chlorella viruses.

An open reading frame (ORF) with strong homology to eukaryotic serine/threonine protein kinases was found in the two Chlorella viruses SC-1A and PBCV-1. The deduced molecular weights of each putative protein kinase were 35 kDa and the predicted amino acid sequences of the two proteins were 95% identical. The ORF encoding the SC-1A protein kinase was over-expressed as a fusion protein in Escherichia coli. The recombinant fusion protein had autophosphorylation activity and could phosphorylate certain exogenous proteins. Antiserum against the recombinant fusion protein reacted with a 35 kDa protein plus three larger proteins from virus infected cells. The 35 kDa protein was a late protein; however, the 35 kDa protein was not packaged in the virion, even though virions contain protein kinase activity.

Amino Acid Sequence↗

Augmentation of the resistance against Escherichia coli by oral administration of a hot water extract of Chlorella vulgaris in rats.

In previous studies, we demonstrated that a hot water extract of Chlorella vulgaris (CVE) augmented the resistance against an intraperitoneal infection with Escherichia coli by its intraperitoneal, intravenous or subcutaneous administration. The augmented resistance appeared to be attributable to the enhanced activity of polymorphonuclear leukocytes (PMN). In this study, the effect of oral administration of CVE against Escherichia coli infection was examined. Male Fisher rats (F344/DuCrj) were administered 1000 mg/kg of CVE orally for 14 days and challenged with 2.7 x 10(8) Escherichia coli intraperitoneally. The numbers of living bacteria in the peritoneal cavity, blood, spleen and liver at 1, 6, and 24 h after the inoculation were counted. The bacterial numbers increased during 1-6 h and reached the peak at 6 h in both control and CVE-administered groups. The bacterial numbers decreased to an undetectable level at 24 h in both groups. In a CVE-administered group, the numbers of viable bacteria in each organ were remarkably lower than those in a control group in all organs so far tested. Whereas, the leukocyte numbers, especially PMN numbers, in the peritoneal cavity and peripheral blood maintained higher levels in the CVE-administered group at 6 h after E. coli inoculation. Chemiluminescent responses of peritoneal exudate cells induced by casein or E. coli were higher in a CVE-administered group. These results form the basis for the judgment that the degree of effectiveness of bacteria clearance from the peritoneal cavity shown by oral CVE administration may be strong enough to warrant developing this material as a new type of biological response modifier.

Administration, Oral↗

Accelerated restoration of the leukocyte number and augmented resistance against Escherichia coli in cyclophosphamide-treated rats orally administered with a hot water extract of Chlorella vulgaris.

The effects of oral administration of a hot water extract of Chlorella vulgaris (CVE) on the restoration of the leukocyte number and on the resistance against Escherichia coli infection were examined in cyclophosphamide (CY)-treated rats. Male Fischer rats (F344/DuCrj) were administered orally 1000 mg/kg of CVE for 14 days and injected intraperitoneally with a single dose of CY (50 mg/kg) (day 0) one day after the 14th CVE administration. CVE was further administered continuously after CY treatment until the rats were sacrificed for analysis. The number of bone marrow cells in the CY + CVE group was significantly higher on day 7 after CY treatment than that in the CY-treated group. The number of spleen cells in the CY + CVE group became significantly higher on day 11 than that in the CY-treated group. In the peripheral blood, the number of PMN recovered efficiently in the CY + CVE group in comparison with the CY-treated group on day 7. When E. coli was injected i.p. into normal, CY-treated, and CY + CVE-treated rats on day 6, the difference in number of bacteria among these three groups was most prominent before 6 h, that is, the number in the CY + CVE group was remarkably lower than those in the CY-treated group, and even in the control group, among all organs so far tested.

Administration, Oral↗

Hot water extracts of Chlorella vulgaris reduce opportunistic infection with Listeria monocytogenes in C57BL/6 mice infected with LP-BM5 murine leukemia viruses.

The bacterial elimination after infection with Listeria monocytogenes was impaired in mice with murine acquired immunodeficiency syndrome (MAIDS) by infection with LP-BM5 murine leukemia virus. Oral administration of hot water extracts of Chlorella vulgaris (CVE) restored the capacity of MAIDS mice to eliminate L. monocytogenes in association with improvement of the deteriorated immune response to L. monocytogenes. DTH response to Listeria in CVE-treated MAIDS mice was significantly higher than that of MAIDS mice after Listeria infection in association with increases in number of CD4+CD8- and CD4-CD8+ alpha beta T-cells in the infected sites. CVE might be effective in the treatment of opportunistic infection in retrovirus-induced immunodeficient patients.

Animals↗

Pilot CELSS based on a maltose-excreting Chlorella: concept and overview on the technological developments.

A typical ecosystem is composed of three compartments: photosynthetic producer (anabolizing processes), consumer and decomposer (catabolizing processes). It is still far too much complex, however, to form the basis on which establishing an engineered artificial ecosystem, dedicated to support life (of the consumer) in space. A simpler, two compartments, pilot model to start with has been selected. It is based on a symbiotic Chlorella (strain 241.80), which can be tuned, at low pH, to produce maltose. This feature prevents the accumulation of useless biomass, not readily edible by the consumer. Being excreted, maltose is easily recoverable, and constitutes a direct source of carbon suitable for many consumers. Since they will totally catabolize it back to CO2, the necessity for a decomposer compartment is avoided. The present status of the technological concept designed to support life of small consumers (animals, microorganisms) will be presented, taking into account the space compatibility of the technologies developed.

Biomass↗

Peculiarities of ultrastructure of Chlorella cells growing aboard the Bion-10 during 12 days.

The ultrastructure of Chlorella cells grown in darkness on a solid agar medium with organic additions aboard the Bion-10 biosatellite was studied. Certain differences in submicroscopic organization of organelles in the experimental cells were revealed compared to the Earth control. The changes are registered mainly in ultrastructure of energetic organelles--mitochondria and plastids of the experimental cells, in particular, an increase of mitochondria and their cristae size, as well as an increase of the total volume of mitochondrion per cell were established. The decrease of the starch amount in the plastid stroma and the electron density of the latter was also observed. In many experimental cells, the increase of condensed chromatin in the nuclei has been noted. Ultrastructural rearrangements in cells after laboratory experiment realized according to the thermogram registered aboard the Bion-10 were insignificant compared to the flight experiment. Data obtained are compared to results of space flight experiments carried out aboard the Bion-9 (polycomponent aquatic system) and the orbital station Mir (solid agar medium).

Chlorella↗