Column chromatographic pre-concentration of iron(III) in alloys and biological samples with 1-nitroso-2-naphthol-3,6-disulphonate and benzyldimethyltetradecylammonium-perchlorate adsorbent supported on naphthalene using atomic absorption spectrometry.

The solid ion-pair material produced from the reaction between benzyldimethyltetradecylammonium chloride (BDTA) and sodium perchlorate on naphthalene provides the basis for a simple, rapid and selective technique for pre-concentrating iron from up to 500 ml of aqueous solution. Iron reacts with disodium 1-nitroso-2-naphthol-3,6-disulphonate (Nitroso-R salt) to form a water-soluble coloured chelate anion. The iron chelate anion forms a water-insoluble, stable iron-Nitroso-R-BDTA complex on naphthalene packed in a column. Trace amounts of iron are quantitatively retained on naphthalene in the pH range 3.5-7.5 and at a flow-rate of 1-2 ml min-1. The solid mass is dissolved out from the column with 5 ml of N,N-dimethylformamide and iron is determined by means of an atomic absorption spectrometer at 248 nm. The calibration graph is linear for concentrations of iron over the range of 0.5-20 micrograms in 5 ml of final solution. The standard deviation and relative standard deviation were calculated. The detection limit of the method was 0.0196 micrograms ml-1 of iron. The sensitivity for 1% absorption was 0.072 microgram ml-1 (0.165 microgram ml-1 by direct atomic absorption spectrometry of aqueous solution). The proposed method was applied to the determination of iron in standard alloys and biological samples.

Immunological identification and quantification of light harvesting chlorophyll a/b protein of Chlorella protothecoides.

The alteration of photosynthetic membrane proteins in relation to the disappearance of pigments during the heterotrophic growth of Chlorella protothecoides was investigated. Chlorophylls and certain polypeptides associated with the LHC II disappeared after 50 hr of heterotrophic growth but the 24 kDa apoprotein constituting LHC II was not affected. Immunological analysis indicated that the chlorophylls and the light harvesting complex proteins of the thylakoid membranes are not tightly coupled and the latter is retained in its native form irrespective of the presence or absence of the former. The circumstantial evidence that the other photosynthetic membrane polypeptides are degraded along with the pigments due to increased proteolytic activity in the rapidly dividing heterotrophic cells indicate that chlorophyll synthesis is not a pre-requisite for the synthesis of the LHC II apoprotein.

Effect of Chlorella vulgaris extracts on murine cytomegalovirus infections.

The host-mediated antiviral effect of Chlorella vulgaris extracts (CVE) was evaluated in ICR mice against murine cytomegalovirus (MCMV) infection. Mice treated with 10 mg of CVE on days 3 and 1 before virus challenge survived lethal infection. The protective effect of CVE was shown by a decrease in the infectious viruses replicated in the target organs of CVE-treated ICR mice. CVE also protected mice from histopathological damage to the target organs due to MCMV infection. Both the serum interferon (IFN) level and 2'5'-oligoadenylate (2-5A) synthetase activity were elevated in CVE-treated and MCMV-infected mice, and were higher than those in the control mice. The natural killer (NK) activity of spleen cells, which is otherwise deteriorated by lethal MCMV infection, was remarkably augmented in CVE-treated mice. Neither virocidal nor virostatic activity of CVE on MCMV was seen in vitro. Thus, CVE-induced resistance against MCMV seems to be host-mediated. The protective effect correlated with the augmentation of NK activity of the spleen cells and serum IFN activity, which inhibited replication of MCMV in the target organs and subsequently saved the mice from death.

Mutual exclusion occurs in a Chlorella-like green alga inoculated with two viruses.

Progeny viruses resulting from dual inoculations with different and near-isogenic viruses of a Chlorella-like green alga were distinguished by immunoblotting. Plaques arising from single cells inoculated with two viruses usually contained only one of the viruses. Thus the viruses mutually exclude one another. In some combinations the ratio of viruses (as infective centres) recovered differed significantly from the input ratio.

Exclusion chromatography with controlled-pore glass beads to isolate Chlorella chromatin and its applications.

A simple and rapid method was developed to isolate chromatin from the unicellular alga, Chlorella, by exclusion chromatography utilizing controlled-pore glass beads. This method takes advantage of the giant size of the chromatin supramolecules and does not require the preliminary isolation of cell nuclei. In order to raise the histone yield, commercially available materials were silanized with dimethyldichlorosilane. The isolated algal chromatin had properties similar to those of other organisms, and the histones contained all five components found in calf thymus. A hierarchy of the higher order structures was also observed in the algal chromatin. This method can be used for the study of chromatin in various cell types, especially in microbial cells, from the viewpoints of not only mere preparation but also cell dynamics and fractionation in relation to the specific components or activities. Some application examples are presented.

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.

Effect of lipophilic extract of Chlorella vulgaris on alimentary hyperlipidemia in cholesterol-fed rats.

The effect of glycolipid (GL) and phospholipid (PL) fractions obtained from Chlorella on serum lipid level and fecal excretion of steroids were examined in cholesterol-fed rats. The increase of the level of serum lipids were inhibited by the feeding of GL, PL and Chlorella powder almost the same degree. Fecal excretion of steroids (mostly of cholesterol, deoxycholic and lithocholic acid) were increased by feeding of GL and PL fractions. It is concluded that the feeding of each fraction inhibits the absorption of exogenous steroids and promotes turnover of bile acids in liver to suppress the increase of serum cholesterol level caused by administration of high cholesterol diet.

Evidence from high-pressure liquid chromatography for the existence of two ferredoxins in plants.

Crude ferredoxin preparations were obtained from blue-green algae, green algae, ferns, and higher plants. We analyzed the preparations by high-pressure liquid chromatography using two different types of columns, a hydrophobic phenyl-5PW column and an ion-exchange DEAE-5PW column. Two ferredoxins were detected in all plants analyzed. The ferredoxins from some plants were separated by use of both columns and those from others were separated by one of the two columns. Thus, there were three possible ways in which pairs of ferredoxins from a single species of plant could be separated. We suspect that there are two different ferredoxins in most if not all species of plants.

The cell cycle of symbiotic Chlorella. IV. DNA content of algae slowly increases during host starvation of green hydra.

The distribution of DNA content of symbiotic Chlorella algae freshly isolated from green hydra was compared with that of cultured Chlorella of the NC64A strain, using flow cytometry. In nonlogarithmic cultures of NC64A most cells had accumulated in G1 phase, while in logarithmic cultures a peak containing cells in S phase and mitosis could be distinguished from the larger G1 peak. However, symbiotic algae showed a single broad peak in which there was no clear distinction between G1 and S phase/mitosis. When hydra were starved for a prolonged period, inhibiting host cell and algal division, the DNA content of the symbiotic algae slowly increased, and the number of daughter cells produced after a single feeding increased with the length of the preceding period of starvation. This suggests that symbiotic algae are able to cycle slowly through S phase, but unless the host is fed they cannot traverse into mitosis and complete the cell division cycle. No significant difference in cell size was found between algae producing either four or eight daughter cells after 1-day- or 22-day-starved hydra were fed, suggesting that algal cell size did not determine the number of daughter cells produced. Instead, this may be dependent upon the length of time the cell had spent in S phase prior to receiving the, as yet unknown, stimulus to enter into mitosis.

Determination of 5-aminolevulinic acid in biological samples by high-performance liquid chromatography.

5-Aminolevulinic acid (ALA), the common precursor of all naturally occurring tetrapyrroles, forms a stable condensation product with 2-amino-3-hydroxynaphthalene which can be identified by its fluorescence. Separation of the compound by reversed-phase high-performance liquid chromatography on RPC-18 columns allows its detection down to the picomolar range and can be successfully applied for ALA analysis in small biological samples. The reaction product of ALA with 2-amino-3-hydroxynaphthalene has been synthesized and characterized.

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.

Hydrocarbons in green and blue-green algae.

Liquid column chromatography and thin-layer chromatography were used to determine the total content of hydrocarbons and gas chromatography was used to evaluate composition of hydrocarbons in green algae (Chlorella kessleri, C. vulgaris, Chlorella sp., Scenedesmus acutus, S. acuminatus, S. obliquus) and the blue-green alga (Spirulina platensis) cultivated under autotrophic or heterotrophic conditions. In C. kessleri cultivated under heterotrophic conditions the content of hydrocarbons was found to be about 10(-2)% (per dry mass), whereas under autotrophic conditions it was about 10(-3)% (per dry mass). The highest content of hydrocarbons was detected in species of the genus Scenedesmus cultivated autotrophically (10(-1)%). Heptadecane and hexacosane were found as major alkanes, 1-heptadecene was detected among alkenes.

[Nuclear RNP containing pre-mRNA. 16. Cross-linked informofers]. / Iadernye RNP, Soderszhashcie pre-mRNK. XVI. "Zashitye" informofery.

The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethylsuberimidate and dimethyl-3,3'dithiobispropionimi-date). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers, being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linking reagent has been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or polyparticles) were reconstituted upon mixing or cross-linked informofers with pre-mRNA and removal of 2 M NaCl. The reconstituted particles were indistinguishable from the original ones by several tests.

Causes of the resistance of Chlorella strains to physical and chemical environmental factors.

Factors have been studied which are responsible for the resistance of cells of several strains of Chlorella to physical and chemical stresses; measurements have been made of the effects of UV and x irradiation, heat treatment, detergents, cadmium salts, and insecticides. Cells which were resistant to the effects of physical factors were also resistant to chemical intoxication. A positive correlation was found between the resistance of cells to the described factors and the level of endogenous thiols in the cells, as well as the free radical content. This in turn is due to an increased number of reaction centers in the electron-transport chain.