COCCOLITH-ASSOCIATED POLYSACCHARIDES FROM CELLS OF EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Coccoliths of Emiliania huxleyi (Lohmann) Hay and Mohler, a unicellular calcifying alga, consist of calcite closely associated with an acidic, Ca²⁺-binding polysaccharide. This polysaccharide is thought to play a regulatory role in coccolith synthesis by interfering with CaCO₃ crystallization. Here we show that the polysaccharides from three different strains, A 92, L and 92 D, all inhibit the precipitation of CaCO₃ in vitro to the same extent. The monosaccharide compositions of the A 92 and L polysaccharide are similar. The 92 D material, however, deviates from the other two: it contains significantly lower amounts of methylated sugars and ribose, and elevated levels of rhamnose and galactose. It also contains antigenic determinants not detected in the A 92 and L polysaccharides. In contrast to the latter two macromolecules the 92 D polysaccharide migrates as two bands upon polyacrylamide gel electrophoresis, possibly resulting from complexing with small amounts of protein. The coccolith polysaccharide from L cells, cultured at an elevated growth rate, also migrates as two bands. This phenomenon is due to an increase in molecular size distribution. The results suggest that certain properties of the molecule may be subject to variation without interfering with its function.     

Three Types of Acidic Polysaccharides Associated with Coccolith of Pleurochrysis haptonemofera: Comparison with Pleurochrysis carterae and Analysis Using Fluorescein-Isothiocyanate-Labeled Lectins

In the coccolithophorid microalgae acidic polysaccharides are considered to be involved in the formation of the calcified scale, coccolith. Characteristics of the acidic polysaccharides extracted from the cell surface of the coccolithophorid Pleurochrysis haptonemofera were analyzed. The acidic polysaccharides on the cell surface can be detected by measuring fluorescence of cells after fluorescein-isothiocyanate-labeled lectin staining by flow cytometry. Flow cytometric analyses revealed that the acidic polysaccharides remained on the cell surface even after CaCO₃ in the coccolith was dissolved by lowering pH, but they were extracted by subsequent EDTA or EGTA treatment, suggesting that they are bound not into the CaCO₃ crystals of the coccolith, but onto the surface via Ca²⁺. Analyses of the acidic polysaccharides by anion exchange chromatography, colloidal precipitation with divalent cations, and polyacrylamide gel electrophoresis (PAGE) revealed that P. haptonemofera has 3 types of acidic polysaccharides (Ph-PS-l, -2, and -3). The PAGE patterns suggested that Ph-PS-2 has a repeated structure with a broad range of molecular weight, as in Pleurochrysis carterae, while Ph-PS-1 and -3 contain several minor components in addition to a major component, respectively. The minor components in Ph-PS-1 and -3 that have not been found in P. carterae might be characteristic of P. haptonemofera. Analyses of both the cell surface treated by various concentrations of EDTA and EGTA and the extracts suggested that Ph-PS-2, which is distinguishable by a higher affinity to concanavalin A, is bound onto the coccolith surface more intensely than the other two types of acidic polysaccharides.     

Polyanion-mediated mineralization — assembly and reorganization of acidic polysaccharides in the Golgi system of a coccolithophorid alga during mineral deposition

Summary Immunolocalization of two highly acidic polysaccharides (PS-1 and PS-2) in a calcifying algaPleurochrysis carterae is described throughout the mineralization process, from before crystal nucleation through the cessation of crystal growth. This unicellular coccolithophorid alga is a useful model for mineralization because it produces calcified scales known as coccoliths in homogeneous cell culture. PS-1 and PS-2 were localized in the crystal coats of mature coccoliths and in electron dense Golgi particles. The polyanions are synthesized in medial Golgi cisternae and co-aggregate with calcium ions into discrete 25 nm particles. Particle-laden vesicles bud from cisternal margins and fuse with a coccolith-forming saccule containing an organic oval-shaped scale which forms the base of the future coccolith. The particles are localized on the base before the onset of mineral deposition and are present in the coccolith saccule throughout the period of crystal (CaCO₃) nucleation and growth. During the final phase of coccolith formation, the particles disappear, and the mature crystals acquire an amorphous coat containing PS-1 and PS-2 polysaccharides which remain with the mineral phase after the coccoliths are extruded from the cell. Postulated mechanisms of polyanion-mediated mineralization are reviewed and their relevance to the calcification of coccoliths is addressed.Abbreviations PS-1 polysaccharide one - PS-2 polysaccharide two - BSA bovine serum albumin - SDS sodium dodecyl sulfate - MES 2-(N-morpholino)-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - DHA 3-deoxy-lyxo-2-heptulosaric acid - TCA trichloroacetic acid     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

CALCIFICATION BY COCCOLITHOPHORIDS: EFFECTS OF pH AND Sr1

Cells of Coccolithus huxleyi which fail to deposit CaCO₃ and form coccoliths often occur as unwanted components in cultures used for studies of calcification. Non-calcified cells generally cannot be made to recalcify, but they can be removed from cultures by treatment at elevated pH or by a method based on faster sinking of calcified cells. Lowering the concentrations of nitrate, phosphate, or trace metals in the medium did not restore calcifying ability of non-calcified cells. However, addition of strontium did promote recalcification of decalcified Cricosphaera carterae grown under calcium limitation. Strontium seemed to promote coccolith attachment to cells rather than to affect calcium uptake or coccolith formation itself.     

North Pacific Late Miocene correlations using microfossils,stable isotopes,percent CaCO3, and magnetostratigraphy

A multidisciplinary approach to stratigraphy based on magnetostratigraphy, stable isotopes, percent CaCO₃ and microfossils provides a framework for paleoclimatic and paleoceanographic reconstruction of the equatorial and North Pacific. A high-resolution biochronologic time scale has been achieved through integration of diatom, radiolaria, coccolith and planktic foraminifer datum levels with direct or indirect correlations to the paleomagnetic time scale. Over 70 datum levels have been identified between 12.5 and 5 Ma from low and middle latitudes resulting in an unprecedented time control. This high-resolution biostratigraphic control combined with stable-isotopic and percent CaCO₃ analyses permits identification of regional as well as global paleoceanographic events.     

Sequential Coccolith Morphogenesis in Hymenomonas carterae

SYNOPSIS. A careful re-examination with refined technics of the ultrastructure of the formation of calcified scales (coccoliths) in the marine unicellular alga Hymenomonas carterae has yielded new and more detailed information about the structure and morphogenesis of these unique and complex Golgi-elaborated organelles. The coccolith rim is formed from 2 distinct, alternating, anvil-shaped elements, 13–16 each, fitted together with a “right-handed” asymmetry. The coccolith is assembled in Golgi cisternae from 2 precursors, a single, scale-like base and multiple granular elements called coccolithosomes. The association of scales and coccolithosomes and subsequent development to the mature coccolith occur in a characteristic sequential fashion within what is one of the better examples of a polarized Golgi. Morphogenesis involves a special cisternal membrane association with the base of the coccolith, the contribution of granular material by coccolithosomes to form the outer rim matrix, and the subsequent filling of the area enclosed by the matrix with an electron-dense material, presumably CaCO₃. A “microenvironment” model system for species specific shape-determination of calcified elements is proposed.     

DISTRIBUTION OF TWO TYPES OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) IN THE NORTHEAST ATLANTIC REGION AS DETERMINED BY IMMUNOFLUORESCENCE AND COCCOLITH MORPHOLOGY1

Culture strains of Emiliania huxleyi (Lohmann 1902) Hay et al. 1967 were placed into two groups designated E. huxleyi type A and type B on the basis of coccolith morphology and immunological properties of the coccolith polysaccharide. We studied the distribution of these types in the North Atlantic region using an indirect immunofluorescence assay with antisera directed against the coccolith polysaccharide of E. huxleyi type A and type B and epifluorescence microscopy. In field samples taken in the Northeast Atlantic Ocean, E. huxleyi type A was found exclusively. In contrast, type B was dominant in the North Sea. Scanning electron microscopy of the samples revealed the same unequal distribution of the two types as found with the immunofluorescent-labelling assay.     

Studies on lectins. XXXVII. Isolation and characterization of the lectin from Jimson-weed seeds (Datura stramonium L.)

The lectin of Jimson-weed seeds (Datura stramonium L.) was isolated by affinity chromatography on a polysaccharide mixture from mycelium of Aspergillus niger. The lectin yields two bands on disc electrophoresis, it has sedimentation coefficient s20,w = 3.8 S and its apparent molecular weight estimated by thin layer gel chromatography is 120,000. The lectin reduced with mercaptoethanol yields on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate three zones corresponding to subunits of molecular weight 72,000, 45,000 and 25,000. The lectin contains large amounts of cystine, glycine, 6.3% of hydroxyproline residues, 4.5% glucosamine and 28% of neutral sugar, predominantly arabinose. The lectin is nonspecific in human erythrocyte ABO system, it is not inhibited by simple sugars but is inhibited by a partial hydrolysate of chitin-containing mixture of polysaccharides from Aspergillus niger.     

Effects of base-soluble proteins and methanol-soluble polysaccharides from corn on mycelial growth of Aspergillus flavus

The antifungal activity of base-soluble proteins (BSP) and methanol-soluble polysaccharides (PS) from A. flavus resistant (Yellow Creole) and susceptible (Huffman) genotypes of corn were investigated by in vitro studies. Bioassays of fungal growth inhibition in agar media showed antifungal activity by proteins and polysaccharides only from the Huffman genotype. Microgramme quantities of protein and polysaccharides were required to retard fungal growth. The polysaccharides have molecular weights greater than 3.5 kilodaltons. Cathodic PAGE of native protein from the two genotypes showed six protein bands with differences in staining intensity of individual components. SDS-PAGE showed four distinct bands in Yellow Creole that were absent in Huffman. Both of the protein samples contained traces of carbohydrate. Analysis of hydrolyzed polysaccharide from the two genotypes showed different proportions of mannose and glucose.     

POLYSACCHARIDES FROM THE ENVELOPES OF HETEROCYSTS AND SPORES OF THE BLUE-GREEN ALGAE ANABAENA VARIABILIS AND CYLINDROSPERMUM LICHENIFORME1

The polysaccharides from the envelopes of heterocysts of Cylindrospermum licheniforme Kütz., and of heterocysts and spores of Anabaena variabilis Kütz., like those from the differentiated cells of Anabaena cylindrica Lemm., have a 1,3-linked backbone consisting of glucosyl and mannosyl residues in a molar ratio of approximately 3:1. As is the case with A. cylindrica the polysaccharides from A. variabilis and from the heterocysts of C. licheniforme have terminal xylosyl and galactosyl residues as side branches. In addition, the polysaccharide from C. licheniforme resembles that from A. cylindrica in having terminal mannosyl residues as side branches (absent from A. variabilis). The polysaccharides from A. variabilis resemble that from A. cylindrica in having glucose-containing side branches (absent from the heterocyst polysaccharide from C. licheniforme), but in contrast to the polysaccharides from the other two species they also have terminal arabinosyl residues as side branches. All of the polysaccharides mentioned appear to be structurally related; we present tentative structures for those not previously investigated. In contrast, the envelope of spores of C. licheniforme contains only a largely 4-linked galactan. The bulk of this envelope is not polysaccharide in nature, and contains aromatic groups.     

On the nature and causes of luminescent lines and bands in coral skeletons

Holes pushed into the surface of laboratory grade CaCO₃ powder reproduced visible and measurable luminescence similar to that seen and measured in coral skeletons. Heating such powder to 450 °C for 2 h did not destroy the luminescence although it did destroy luminescence in powdered coral skeleton. The effect in coral skeletal powder was probably due to carbonisation of contained organics because addition of small and increasing amounts of powdered charcoal to laboratory grade CaCO₃ increasingly attenuated luminescence. Luminescent lines and bands in coral skeletons have previously been ascribed to incorporation of humic substances. However, coating laboratory grade powder with humic acid attenuates rather than enhances luminescence. Ultra-violet lamps used to display coral luminescent lines and bands emit significant amounts of violet and blue visible light. Reflection of these visible wavelengths from the surface of laboratory grade CaCO₃ powder obscured luminescence of the powder. Multiple reflections within a hole in the powder resulted in absorption of the short wavelengths of visible light, including violet and blue light that would otherwise mask luminescence, and their re-emission at longer wavelengths. Luminescent bands in offshore corals were associated with the low-density regions of the annual density banding pattern. Luminescent lines in skeletons of inshore corals were in narrow regions of low-density skeleton, probably resulting from altered growth during periods of lowered salinity. Accepted: 20 April 2000     

Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains

Production of lactic acid from paper sludge was studied using thermophilic Bacillus coagulan strains 36D1 and P4-102B. More than 80% of lactic acid yield and more than 87% of cellulose conversion were achieved using both strains without any pH control due to the buffering effect of CaCO₃ in paper sludge. The addition of CaCO₃ as the buffering reagent in rich medium increased lactic acid yield but had little effect on cellulose conversion; when lean medium was utilized, the addition of CaCO₃ had little effect on either cellulose conversion or lactic acid yield. Lowering the fermentation temperature lowered lactic acid yield but increased cellulose conversion. Semi-continuous simultaneous saccharification and co-fermentation (SSCF) using medium containing 100 g/L cellulose equivalent paper sludge without pH control was carried out in serum bottles for up to 1000 h. When rich medium was utilized, the average lactic acid concentrations in steady state for strains 36D1 and P4-102B were 92 g/L and 91.7 g/L, respectively, and lactic acid yields were 77% and 78%. The average lactic acid concentrations produced using semi-continuous SSCF with lean medium were 77.5 g/L and 77.0 g/L for strains 36D1 and P4-102B, respectively, and lactic acid yields were 72% and 75%. The productivities at steady state were 0.96 g/L/h and 0.82 g/L/h for both strains in rich medium and lean medium, respectively. Our data support that B. coagulan strains 36D1 and P4-102B are promising for converting paper sludge to lactic acid via SSCF.     

Production of exopolysaccharide from ethanol inAgrobacterium radiobacter

Agrobacterium radiobacter produces an extracellular polysaccharide from various carbon sources. The exopolysaccharide is produced from ethanol also in a minimal medium containing nitrate as the sole nitrogen source. On cultivation in a medium without CaCO₃ only a minute amount of ethanol is converted to the exopolysaccharide. Both ethanol and nitrate in higher concentrations inhibit the growth rate. In a medium with CaCO₃ the proportion of ethanol converted to the polysaccharide is about ten-fold higher and the inhibitory effect of ethanol and nitrate on the growth rate is analogous to that found in the medium without CaCO₃-Comparison of results of mass and electron balance with a kinetic model shows that the parameters obtained in cultivations with CaCO₃ are less reliable. The balances point to the possibility of formation of other extracellular products.     

Polysaccharide composition of unlignified cell walls of pineapple [Ananas comosus (L.) Merr.] fruit.

The polysaccharides of cell walls isolated from the fleshy, edible part of the fruit of the monocotyledon pineapple [Ananas comosus (L.) Merr.] (family Bromeliaceae) were analyzed chemically. These cell walls were derived mostly from parenchyma cells and were shown histochemically to be unlignified, but they contained ester-linked ferulic acid. The analyses indicated that the noncellulosic polysaccharide composition of the cell walls was intermediate between that of unlignified cell walls of species of the monocotyledon family Poaceae (grasses and cereals) and that of unlignified cell walls of dicotyledons. Glucuronoarabinoxylans were the major non-cellulosic polysaccharides in the pineapple cell walls. Xyloglucans were also present, together with small amounts of pectic polysaccharides and glucomannans (or galactoglucomannans). The large amounts of glucuronoarabinoxylans and small amounts of pectic polysaccharides resemble the noncellulosic polysaccharide composition of the unlignified cell walls of the Poaceae. However, the absence of (1-->3,1-->4)-beta-glucans, the presence of relatively large amounts of xyloglucans, and the possible structure of the xyloglucans resemble the noncellulosic polysaccharide composition of the unlignified cell walls of dicotyledons.     

Chelating agents improve enzymatic solubilization of pectinaceous co-processing streams

This study investigates the hypothesis that loosening of the egg-box structure by presence of divalent ion chelating agents during enzymatic degradation of homogalacturonan (HG) can improve enzymatic polysaccharide solubilization on pectinaceous, agro-industrial co-processing streams. The influence of different levels of ethylene-diaminetetraacetic acid (EDTA), citric acid, oxalic acid, and phosphate was assessed in relation to enzymatic solubilization of isopropanol precipitatable oligo- and polysaccharides from sugar beet pulp, citrus peel, and two types of potato pulp. The two types of potato pulp were FiberBind 400, a dried commercial potato pulp product, and PUF, a dried calcium reduced product, respectively. The enzymatic treatment consisted of 1% (w/w) of substrate treated with pectin lyase from Aspergillus nidulans and polygalacturonase from A. aculeatus [each dosed at 1.0% (w/w) enzyme/substrate] at 60 °C, pH 6.0 for 1 min. Characterization of the released fractions demonstrated a significantly improved effect of chelating agents for polysaccharide solubilization from FiberBind 400, PUF, and citrus peel, whereas only low amounts of polysaccharides were solubilized from the sugar beet pulp. The results substantiated the importance of chelating agents during enzymatic extraction of pectinaceous polysaccharides. Lower levels of chelating agents were required for the calcium-reduced potato pulp substrate (PUF) indicating the significance of calcium cross-linking in HG in relation to the enzymatic solubilization yields. The effect of the chelating agents correlated to their dissociation constants (pK_a values) and calcium binding constants and citric acid and EDTA exerted highest effects. Maximum polysaccharide yield was obtained for FiberBind 400 where the enzymatic treatment in presence of citric acid yielded 22.5% (w/w) polysaccharides of the initial substrate dry matter.     

Optimization of renewal regime for improvement of polysaccharides production from Porphyridium cruentum by uniform design

To probe the effects of renewal regime on the production of polysaccharides, Porphyridium cruentum was cultured semi-continuously in flat plate photobioreactor. Uniform design was used to optimize renewal conditions. Quadratic mathematic models related to productivity, total recovery yield of biomass and polysaccharides were set up to clarify the influence of individual factors and their interactions. According to the mathematic models, the optimal semi-continuous condition for total yield of polysaccharide was NaNO₃ 3.5 g/L, renewal rate 27%, renewal period 2.91 days. The optimal condition for polysaccharide output rate was NaNO₃ 0.5 g/L, renewal rate 5%, renewal period 7 days. With the optimal renewal regime, the maximal total recovery yields of polysaccharide achieved at 29.4 g, which was 1.57 times higher than that of batch cultivation. The maximum output rate of polysaccharide was 68.64 mg/L per day, which was 2.02 times higher than previous reported data.     

Scavenging and antioxidant activities of immunomodulating polysaccharides isolated from Salvia officinalis L.

Crude polysaccharides, isolated from the aerial parts of sage (Salvia officinalis L.) by sequential extraction with water (A), hot ammonium oxalate (B), dimethyl sulfoxide (C), 1 M (D) and 4 M (E) potassium hydroxide solutions, and six ion-exchange fractions of A were examined for their ability to inhibit peroxidation of liposome lipid by hydroxyl radicals and to reduced DPPH radical content. The highest inhibition of liposome lipid peroxidation was found with crude polysaccharides A, B and D, antioxidant activities reached ～37%. The purified fractions A1 and A2 inhibited the liposome peroxidation to ～35%. However, the radical scavenging abilities of the most active crude polysaccharides A, B and C on DPPH radicals were found in the range 80–90%, while the most active purified fractions A₃–A₆ in three or fourfold doses achieved 75–92%. The least effective tested polysaccharides succeeded 20% inhibition using both methods.     

Optimization of aqueous-assisted extraction of polysaccharides from pumpkin (Cucurbita moschata Duch) and their biological activities

The pumpkin pulp contains a greater composition of edible polysaccharides and has reported with excellent biological applications. This research pertains to optimize the extraction of polysaccharides from the fleshy portion of the pumpkin using aqueous assisted extraction (AAE). The result showed that the optimal extraction condition of pumpkin polysaccharide was as follows: extraction temperature at 55 °C, pH 4.5, and enzyme concentration of 4000 µ/g for 80 min. Under the optimal extraction condition, the yield of pumpkin polysaccharide via AAE (15.4) was significantly higher. The biological activities of extracted polysaccharide including α-amylase inhibition (57.41% at 1000 µg/mL) and anti-inflammatory (50.41% at 25 µg/mL) activity increased significantly. Additionally, the antioxidant activities of extracted pumpkin polysaccharides including IC₅₀ values of DPPH and ABTS were 59.87% and 58.74%, respectively. The pumpkin polysaccharide has maximum inhibitory effects against bacterial strains especially for Escherichia coli than that of fungal strains. It is suggested that the aqueous assisted extraction of is a cost-effective promising method to decrease the processing time as well as enhancing extracted polysaccharide yield – times.     

The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.