Structural features of the sulphated polysaccharide from a green seaweed, Spongomorpha indica.

A sulphated heteropolysaccharide, [alpha]D +59 degrees, was isolated from a green seaweed, Spongomorpha indica, by extraction with ammonium oxalate. The polymer is composed of arabinose, xylose, galactose and glucose in the ratio 8.9:1.0:12.0:1.0. Studies showed that the polysaccharide is a complex and multilinked polymer containing arabinose in both furanose and pyranose forms. The core of the polysaccharide is composed of 1,4-linked galactose units. The arabinofuranose units are present as non-reducing end units, as well as jointed through 1,3- and 1,2-linkages. The majority of the arabinopyranose units are joined through 1,4-linkages. Xylose is present as a branch terminating unit. Glucose is joined through 1,4-linkages. Both arabinose and galactose carry branches. Sulphate groups are present on some of the arabinose units at C-2 and on some of the galactose units at C-2 and C-3.

Structural features of the sulphated polysaccharide from a green seaweed, Cladophora socialis.

A sulphated heteropolysaccharide, [alpha]27D + 59.9 degrees, has been isolated from a green seaweed, Cladophora socialis, by extraction with dilute acid and purified by fractional precipitation. The polymer is composed of galactose (58.3%), arabinose (31.8%), xylose (10.6%) and sulphate (16.9%). The results of methylation analysis, periodate oxidation and partial acid hydrolysis studies indicate that the polymer is a branched one and is composed of 1,3-linked galactose and 1,4-linked arabinose units. Xylose is present at the non-reducing end position of the branches. Both arabinose and galactose carry branches. Desulphation and subsequent analysis of the polymer show that some of the arabinose units carry sulphate groups at C-3 and some of the galactose units carry the sulphate groups at C-4 and some at C-4 and C-6 as well.

The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae).

The sexuality-inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS-22 and separated by reverse-phase HPLC into several isoforms which differ in the degree of O-glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N-glycans which are clustered in the middle of the polypeptide chain. The N-glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gaschromatographic sugar analysis and two-dimensional HPLC separation to possess the following definite structures: (A) Man beta 1-4GlcNAc beta 1-4GlcNAc; (B) (Man alpha)3 Man beta 1-4GlcNAc beta 1-4GlcNAc Xyl beta; (C) (Man alpha)2 Man beta 1-4GlcNAc beta 1-4GlcNAc; (D) (Man)2Xyl(GlcNAc)2. Xyl beta Two of the three N-glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N-glycosylation site. The O-glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.

Plant antimutagenic agents, 7. Structure and antimutagenic properties of cymobarbatol and 4-isocymobarbatol, new cymopols from green alga (Cymopolia barbata).

Two new compounds, cymobarbatol and 4-isocymobarbatol, were isolated from the marine alga Cymopolia barbata. The complete structures and absolute stereochemistries of these compounds were elucidated by a variety of spectroscopic techniques and X-ray crystallography. Both compounds were found to be nontoxic over a broad concentration range to Salmonella typhimurium strains T-98 and T-100. Both compounds exhibited strong inhibition of the mutagenicity of 2-aminoanthracene and ethyl methanesulfonate toward, respectively, the T-98 strain plus a metabolic activator and T-100.

Identification of 3-deoxy-manno-2-octulosonic acid, 3-deoxy-5-O-methyl-manno-2-octulosonic acid and 3-deoxy-lyxo-2-heptulosaric acid in the cell wall (theca) of the green alga Tetraselmis striata Butcher (Prasinophyceae).

The main constituent of the cell wall complex carbohydrate of the scaly green alga Tetraselmis striata Butcher is shown to be 3-deoxy-manno-2-octulosonic acid (42%). In addition two other 2-keto-sugar acids are present, namely, 3-deoxy-5-O-methyl-manno-2-octulosonic acid (7%), the first methylated derivative of 3-deoxy-manno-2-octulosonic acid found in nature, and 3-deoxy-lyxo-2-heptulosaric acid (11%). The characterization of the three 2-keto-sugar acids has been carried out on the corresponding methyl ester methyl glycosides using GLC-MS and 500-MHz 1H-NMR spectroscopy, and on the corresponding reduced alditol acetates using GLC-MS. Other monosaccharides occurring in the cell wall are D-galacturonic acid (14%), D-galactose (4%), D-gulose (2%), D-glucose (1%) and L-arabinose (1%).

A convenient procedure for the isolation of intact translatable mRNA by potassium iodide gradient centrifugation.

High concentrations of KI were found to efficiently protect RNA against degradation by RNases. When a sufficient amount of solid KI was added to cell lysates or subcellular fractions (9 g per 10 ml), the solutions could be stored at room temperature for several days without measurable degradation of mRNA. Ribonucleic acids were selectively sedimented when these KI-containing solutions were centrifuged at 72,000 x g for 24 h. The RNA pellets were found to be readily dissolved in bidistilled water and the redissolved RNA could be immediately submitted to oligo(dT)-cellulose chromatography to isolate the poly(A)-containing RNA. However, extraction with phenol/chloroform was found to be necessary, if total RNA or poly(A)-minus RNA was to be analysed. This procedure was found to be superior to other methods currently in use - especially with respect to the isolation of intact, translatable high-molecular-weight mRNA.

A phosphodiester bridge between two arabinose residues as a structural element of an extracellular glycoprotein of Volvox carteri.

The sulphated glycoprotein SSG 185 is the monomeric precursor of a highly aggregated structural element in the extracellular matrix of the multicellular green alga Volvox carteri. A phosphodiester of arabinose was isolated from a saccharide fragment of SSG 185. The structure of this phosphodiester was investigated by methylation analysis, 13C-NMR, photometric methods and enzymatic assays and identified as D-Araiota-5-phospho-5-D-Araiota. The function of this phosphodiester bridge as a crosslink of different carbohydrate chains in SSG 185 is discussed.

Population consequences of cadmium toxicity in soil microarthropods.

Chronic toxicity experiments were performed using the collembolan Orchesella cincta (L.) and the oribatid mite Platynothrus peltifer (Koch), which were exposed to various levels of cadmium in the food (green algae). Experimental results were combined with life-history information to obtain realistic estimates of the intrinsic rate of population increase and its sensitivity to Cd stress. Chronic LC50 values for dietary exposure to Cd were estimated as 1.60 mumol/g for O. cincta and 3.18 mumol/g for P. peltifer. No-observed-effect concentrations (NOECs) for growth and reproduction were 0.042 mumol/g for O. cincta and 0.026 mumol/g for P. peltifer. The main effects of Cd were, however, different in each species. In O. cincta, Cd affected primarily female growth, without a direct effect on reproduction; in P. peltifer, the effect was primarily on reproduction. Uptake of Cd was higher in P. peltifer than in O. cincta, and caused a loss of zinc in the former species. As a consequence of their differing physiological responses to Cd, mites and collembolans also reacted differently in their population growth rates. The capacity for population increase of mites appeared to be rather sensitive to Cd, while collembolans were able, to some extent, to maintain their capacity for increase, in spite of toxic effects at the individual level. These results may contribute to developing ecotoxicological theory for the population consequences of toxic action in species with diverging life histories. Soil microarthropods may be suitable test organisms for evaluating the risk of chemicals to the soil ecosystem.

Immunomodulation by a unicellular green algae (Chlorella pyrenoidosa) in tumor-bearing mice.

A unicellular algae, Chlorella pyrenoidosa, was used as a biological response modifier. In C57BL/6(B6), C3H/He and DDD/1 mice, both intraperitoneal or oral administrations of autoclaved Chlorella cells or heat-extracted substance were carried out every other day for 10 days before mouse mammary carcinoma (MM-2) or Ehrlich ascites cells were transplanted into the peritoneal cavity. In case of mouse leukemia cells (EL-4), subcutaneous transplantation was carried out. All control mice died within 20 days after each tumor cell transplantation, while 73.3-80% of the treated groups survived over 60 days in the combination of MM-2 vs. C3H/He and EL-4 vs. B6, respectively. The cytotoxic activities against tumor cells, that were abolished by treatment with anti-Thyl.2 monoclonal antibody plus complement, were evidenced in the experimental host. Since Chlorella cells and derivatives showed no indication of direct in vitro cytotoxicity to either tumor or mouse spleen cells, the antitumor effects documented may be mediated by host immune response.

Purification and characterization of pentagalloylglucose, and alpha-glucosidase inhibitor/antibiotic from the freshwater green alga Spirogyra varians.

An alpha-glucosidase inhibitor/antibiotic was purified from the freshwater green alga Spirogyra varians and was determined to be the pentagalloylglucose 3-O-digalloyl-1,2,6-trigalloylglucose.

A method for RNA isolation from marine macro-algae.

Sulfated, carboxylic polysaccharides and polyphenols found in marine macro-algae interfere with RNA isolation from these plants and inhibit RNA activities in vitro. Methods based on differential precipitation of RNA or carbohydrates in high salts were used to eliminate the acidic carbohydrates. To protect RNA from inactivation by oxidized polyphenols, strong reducing reagents were used to prevent polyphenol oxidation. RNA was successfully isolated from Macro-cystis pyrifera (brown alga), Porphyra schizophylla (red alga), and Enteromorpha intestinalis (green alga). mRNA isolated from the total RNA was shown to be translationally active.

1H NMR studies of plastocyanin from Scenedesmus obliquus: complete sequence-specific assignment, secondary structure analysis, and global fold.

Two-dimensional 1H NMR methods have been used to make sequence-specific resonance assignments for the 97 amino acid residues of the plastocyanin from the green alga Scenedesmus obliquus. Assignments were obtained for all backbone protons and the majority of the side-chain protons. Spin system identification relied heavily on the observation of relayed connectivities to the backbone amide proton. Sequence-specific assignments were made by using the sequential assignment procedure. During this process, an extra valine residue was identified that had not been detected in the original amino acid sequence. Elements of regular secondary structure were identified from characteristic NOE connectivities between backbone protons, 3JHN alpha coupling constant values, and the observation of slowly exchanging amide protons. The protein in solution contains eight beta-strands, one short segment of helix, five reverse turns, and five loops. The beta-strands may be arranged into two beta-sheets on the basis of extensive cross-strand NOE connectivities. The chain-folding topology determined from the NMR experiments is that of a Greek key beta-barrel and is similar to that observed for French bean plastocyanin in solution and poplar plastocyanin in the crystalline state. While the overall structures are similar, several differences in local structure between the S. obliquus and higher plant plastocyanins have been identified.

Green algal cytochrome b6-f complexes: isolation and characterization from Dunaliella saline, Chlamydomonas reinhardtii and Scenedesmus obliquus.

Cytochrome b6-f complexes have been isolated from Chlamydomonas reinhardtii, Dunaliella saline and Scenedesmus obliquus. Each complex is essentially free of chlorophyll and carotenoids and contains cytochrome b6 and cytochrome f hemes in a 2:1 molar ratio. C. reinhardtii and S. obliquus complexes contain the Rieske iron-sulfur protein (present in approx 1:1 molar ratio to cytochrome f) and each catalyzes a DBMIB- and DNP-INT-sensitive electron transfer from duroquinol to spinach plastocyanin. Immunological assays using antibodies to the peptides from the spinach cytochrome complex show varying cross-reactivity patterns except for the complete absence of binding to the Rieske proteins in any of the three complexes, suggesting little structural similarity between the Rieske proteins of algae with those from higher plants. One complex (D. salina) has been uniformly labeled by growth in NaH14CO3 to determine stoichiometries of constituent polypeptide subunits. Results from these studies indicate that all functionally active cytochrome b6-f complexes contain four subunits which occur in equimolar amounts.

Preparation of tubulin from Caulerpa, a marine green alga, using casein as a protective agent against proteolytic degradation.

Bidirectional organelle movements taking place in the cytoplasm of the rhizomes of Caulerpa, a coenocytic marine green alga, have been indicated to be dependent on microtubules (Kuroda, K. & Manabe, E. (1983) Proc. Jpn. Acad. 59B, 131-134; Manabe, E. & Kuroda, K. (1984) Proc. Jpn. Acad. 60B, 118-121). However, when a crude extract of Caulerpa rhizomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting with monoclonal anti-tubulin antibody, no reacting band could be detected. This apparent absence of tubulin in the extract was found to be a result of the complete degradation of tubulin by potent intrinsic proteolytic activity. All of the commercially available protease inhibitors so far tested (p-chloromercuriphenylsulfonic acid, phenyl methylsulfonyl fluoride, 1-chloro-4-phenyl-3-tosylamido-2-butanone, 7-amino-1-chloro-3-tosylamido-2-heptanone, p-tosyl-L-arginine methyl ester, soybean trypsin inhibitor, antipain, chymostatin, leupeptin, and pepstatin) failed to inhibit the activity completely. But addition of casein at the concentration of 1% (weight per volume) to the solutions used for preparation was effective in protecting tubulin from proteolytic degradation, thus making it possible to prepare tubulin from the crude extract of Caulerpa. On SDS-PAGE, the Caulerpa alpha-tubulin thus prepared was a little smaller in molecular weight than that of rabbit brain.

Photochemistry in the isolated Photosystem II reaction-centre core complex.

The photochemistry of the isolated Photosystem II reaction-centre core from pea and the green alga Scenedesmus was examined by e.s.r. Two types of triplet spectrum were observed in addition to the spin-polarized reaction-centre triplet previously identified. The additional triplet formed on continuous illumination at 4.2 K was attributed to a monomeric phaeophytin molecule. The second triplet, which was stable in the dark at 4.2 K following illumination, was assigned to the radical pair Donor+I-. This provides evidence that an electron donor to chlorophyll P680 is present on the polypeptide D1-polypeptide D2-cytochrome b-559 core complex.

Isoenzyme analysis of lichen algae in immobilized pH gradients.

A base for a modern species' concept of chlorococcal algae can be obtained not by morphological analysis, but by biochemical characters, e.g. isoenzyme banding patterns. From isolated lichen algae of the genus Trebouxia de Puymaly a set of five such enzymes has been studied by isoelectric focusing in immobilized pH gradients (IPG): phosphoglucomutase, phosphoglucose isomerase, malate dehydrogenase, mannitol dehydrogenase and leucine aminopeptidase. The first four are resolved into isoforms in a pH 4-7 IPG interval, while the last one is analyzed in an IPG pH 3.5-5 span. The patterns are specific for distinct populations, inter- and intraspecifically varying in dependence from their geographical distribution or the lichen species from which they have been isolated. Their limited heterogeneity (one to four isoforms) suggests that they are the products of specific genes rather than artefacts of the extraction procedure or the IPG analysis. Sharp isozyme patterns can only be obtained in a mixed-bed, carrier-ampholyte (CA)-IPG gel and by anodic application, suggesting that the recently proposed mechanism of hydrophobic protein-IPG matrix interaction (Electrophoresis, 1987, 8, 62-70) is fully operative here. As an additional mechanism, it is proposed that, in some cases, CA might simply act, when added to an IPG gel, by buffering, in the transient state, the sample zone before the protein migrates from the liquid phase into the IPG matrix.

The partial purification of a factor from Dunaliella salina that causes the rapid in situ inactivation of light-activated chloroplast coupling factor 1 (CF1).

A factor having the expected properties of the in vivo oxidant responsible for inactivating the in vivo light-activated chloroplast coupling factor 1 (CF1) has been partially purified from cell-free extracts of Dunaliella salina. This factor is highly polar, weakly acidic, and relatively temperature stable. The ability of this factor to inactivate light-activated CF1 is prevented if it is pretreated with reductants such as dithiothreitol. The factor has virtually no effect on the ethanol-induced, Mg2+-dependent ATPase activity of the isolated CF1.

H blood group detection by the L-fucose binding lectin of the green marine alga Ulva lactuca.

Extracts of the green marine alga Ulva lactuca collected along the seashore of Tel-Aviv exhibit hemagglutinating activity towards papain-treated human erythrocytes. This hemagglutinating activity was shown to be inhibited by L-fucose and EDTA, and to be relatively resistant to heating at 60 degrees C, while sensitive to low pH. Like the lectin of Ulex europeus, the Ulva lectin exhibits blood group H specificity. It agglutinates most strongly erythrocytes of blood group 0(H) followed by B greater than A greater than AB. A2 and A2B erythrocytes are agglutinated by it considerably more strongly than A1 and A1B respectively. Bombay 0(hh) type erythrocytes are almost non-reactive. The lectin can be stored at -20 degrees C for years.

The use of fluorochromes in the cytochemical characterization of some phytoflagellates.

Sixteen fluorochromes were tested for the cytochemical characterization of two dinoflagellates (Amphidinuim carterae, Prorocentrum micans) and one chlorophycean flagellate (Dunaliella tertiolecta). Depending on the fluorochrome used, various cellular components (including the plasma membrane, thecal plates, pusule, trichocysts, nucleus, lipid bodies and vacuoles) were revealed. The different colours obtained from single or double fluorochrome staining enabled the differentiation and identification of most cellular components. Protoplasmic staining with Fluorescein diacetate suggested the occurrence of esterases in the three phytoflagellates. Rhodamine B, Neutral Red, FluoroBora P and Nile Blue revealed extensive occurrence of lipoid bodies in A. carterae, but Nile Blue showed considerable difference from the other stains in the inclusion size and intracellular location of these bodies. Chlortetracycline binding, and its inhibition by the Ca2+ionophore A23187, indicated that the plasma membrane, pusule system and trichocysts contain sites of Ca2+ binding. Calcofluor White ST proved superior to Congo Red and Lucifer Yellow in elucidating structural details of the thecal plates of P. micans. While Acridine Orange revealed the presence of surface-coat acidic polysaccharides, the fluoresceinated lectins established their glycoconjugate nature in all the three flagellates. Possible mechanisms of fluorochrome uptake are discussed.