Fucans, but not fucomannoglucuronans, determine the biological activities of sulfated polysaccharides from Laminaria saccharina brown seaweed

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed.     

REACTIVE OXYGEN PRODUCTION AND DAMAGE IN INTERTIDAL FUCUS SPP. (PHAEOPHYCEAE)

The research described in this paper was designed to test the hypothesis that the differential stress tolerance associated with the vertical zonation of intertidal seaweeds is attributable to reactive oxygen metabolism. To do so, we measured the production of, and damage caused by, reactive oxygen in three species of intertidal brown seaweeds— Fucus spiralis L., F. evanescens C. Ag., and F. distichus L.— that differ in their ability to withstand freezing, desiccation, and high light stress. Fucus spiralis is the most stress-tolerant species and F. distichus the least. Reactive oxygen production was determined by measuring the production of H₂O₂ and the oxidation of dichlorohydrofluorescein diacetate to dichlorofluorescein. Damage caused by freezing, desiccation, and high-light stress was assessed by measuring variable fluorescence (F_v/F_m) and lipid peroxidation. Production of reactive oxygen increased following freezing, desiccation, or high-light stress. In general, the data were consistent with the hypothesis that reactive oxygen metabolism is involved in stress tolerance. The production of reactive oxygen was relatively low in unstressed seaweeds, and there was little difference between species. Fucus distichus showed the greatest increase in reactive oxygen production after desiccation and freezing stress. Fucus evanescens produced more reactive oxygen production after desiccation than F. spiralis. Although F. evanescens and F. spiralis produced similar amounts of reactive oxygen after freezing, this treatment resulted in an increase in lipid peroxidation only in F. evanescens (and F. distichus ).     

Screening of marine bacteria for fucoidan hydrolases

Twenty-five strains of epiphytic marine bacteria isolated from the brown algae Fucus evanescens and Chorda filum and fifty-three bacteria isolated from the sea cucumber Apostichopus japonicus were screened for fucoidanases using fucoidans prepared from the brown algae F. evanescens, Laminaria cichorioides, and L. japonica. Eighteen bacterial epiphytes and thirty-eight bacterial isolates from the sea cucumber were found to contain fucoidanases, which were able to hydrolyze either all of the fucoidans studied or some of them. Bacteria of the genera Cytophaga and Alteromonas/Pseudoalteromonas exhibited the highest fucoidanase activities, which, however, did not exceed the activity of fucoidanases from the already known sources.     

ATLANTIC KELP SPECIES LAMINARIA LONGICRURIS AND L. SACCHARINA (LAMINARIALES) ARE CONSPECIFIC

The identification of two Atlantic kelp species, Laminaria longicruris and L. saccharina , has been controversial. In order to know if these two species are conspecific, plastid-encoded RuBisCo spacer and nuclear rDNA ITS sequences were analyzed from twelve individuals of L. longicruris , five of L. saccharina , from Connecticut and Nova Scotia. Four individuals of L. digitata from the above coasts were also analyzed as reference in this study. All RuBisCo spacer sequences from L. longicruris and L. saccharina were exactly identical except for one individual with three different nucleotides. Zero to five different nucleotides of ITS sequences, inculding four polymorphic sites, were found in two species. However, their RuBisCo spacer and ITS sequences are quite different from those of L. digitata. These results strongly suggest that L. saccharina (L.) Lamour. 1813 is conspecific with L. longicruris De La Pylaie 1824 and the different local populations, despite various morphotypes, may be genetically very similar.     

REACTIVE OXYGEN METABOLISM IN INTERTIDAL FUCUS SPP. (PHAEOPHYCEAE)

Our previous research suggests that interspecific variation in stress tolerance in intertidal Fucus spp. (Phaeophyceae) is partially mediated by differences in the production of, or ability to detoxify, reactive oxygen. Here we report on the content of antioxidants (ascorbate, glutathione, carotenoids, and tocopherols) and protective enzymes (catalase, superoxide dismutase, ascorbate peroxidase, and glutathione reductase) involved in reactive oxygen metabolism in three species of intertidal brown algae— Fucus spiralis L., F. evanescens C. Ag., and F. distichus L.—that differ in stress tolerance and position in the intertidal zone. Contents of the major antioxidants were similar in the three species and were not correlated with stress tolerance. The least stress tolerant species, F. distichus, had the lowest activity of reactive-oxygen-scavenging enzymes, although F. spiralis, the species with the highest stress tolerance, and F. evanescens contained similar activities of antioxidant enzymes on a fresh-weight basis. However, the activities of superoxide dismutase and ascorbate peroxidase in F. evanescens are lower than those of F. spiralis when expressed on the basis of chlorophyll. These data show that the ratio between reactive oxygen protection and production might be more important than the absolute content of antioxidants and protective enzymes. It also shows the importance of localization of detoxifying mechanisms and avoidance of oxidative stress.     

The Effects of Polyphenolic Impurities in Fucoidan Samples from the Brown Alga Fucus distichus subsp. evanescens (C. Agardh) H.T. Powell, 1957 on the Embryogenesis in the Sea Urchin Strongylocentrotus intermedius (A. Agassiz, 1864) and on the Embryotoxic Action of Cucumarioside

Russian Journal of Marine Biology - The effects of polyphenolic impurities on the biological activity of fucoidan samples from the brown alga Fucus distichus subsp. evanescens were studied using...     

Anticoagulant activity of fucoidans from brown algae

The anticoagulant activity of polysaccharide fucoidans from 11 species of brown algae was studied. The anticoagulant activity was measured by the activated partial thromboplastin time (APTT), prothrombin time, and thrombin time. Inhibitory action of these fucoidans significantly varied from one species to another. Fucoidans from Laminaria saccharina and Fucus distichus exhibited high anticoagulant activity, while fucoidans from Cladosiphon okamuranus and Analipus japonicus were almost inactive. Other fucoidans exhibited intermediate inhibitory activity. The inhibitory effect of fucoidans on thrombin and factor Xa was investigated in the presence or in the absence of natural thrombin inhibitor, antithrombin III (AT III). In contrast to the best-studied anticoagulant, heparin, most of these fucoidans inhibited thrombin in the absence of AT III. In the presence of AT III the inhibitory effect of fucoidans considerably increased. In contrast to heparin, fucoidans weakly influenced factor Xa activity in the presence of AT III and their inhibitory effect was not observed in the absence of AT III. There was no correlation between the anticoagulant activities of this series of fucoidans and their anti-inflammatory action, studied earlier. It is suggested that these two types of fucoidan activities depend on different structural features of fucoidans. Results of this study demonstrate a possibility of preparation of fucoidans with high anti-inflammatory activity but low anticoagulant activity. Anticoagulant activity of the fucoidans did not exhibit direct dependence on the content of fucose, the other neutral sugars and sulfates; no dependence was also found between the anticoagulant activity and the structure of the backbone of their molecules.     

Expressed sequence tag-derived polymorphic SSR markers for Fucus serratus and amplification in other species of Fucus

The seaweed genus Fucus is a dominant component of intertidal shores throughout the North Atlantic and North Pacific and has been the focus of considerable developmental, ecological, and evolutionary research for the past century. Here, we present details of 21 expressed sequence tag-derived simple sequence repeat markers (microsatellites). All 21 were polymorphic for F. serratus, which also display considerable cross-reactivity with the sister species F. distichus (18) and the more distantly related F. vesiculosus (13), and F. spiralis (5).     

KARYOGAMY BLOCK BY HEAT STRESS IN THE FERTILIZATION OF BROWN ALGAE

Karyogamy was inhibited by heat stress in zygotes of Scytosiphon lomentaria (Lyngbye) Link (isogamy), Cutleria cylindrica Okamura (anisogamy), and Fucus distichus subsp. evanescens (C. Agardh) Powell (oogamy). Although high temperatures did not inhibit migration of the male and female nuclei, nuclear envelope fusion was blocked. The ultrastructural stage at which karyogamy was inhibited varied among these species. In S. lomentaria, the outer membranes fused with each other, but the inner membranes did not fuse. Partial fusion of the nuclear envelope occurred in C. cylindrica. In F. distichus, the block of karyogamy at high temperature was incomplete, and nuclear fusion proceeded gradually. The block to karyogamy in S. lomentaria zygotes was reversible, and karyogamy proceeded when zygotes were transferred from 22° to 14° C. Experiments using inhibitors suggested that proteins that might be formed de novo after fertilization do not participate in karyogamy or its inhibition at either 14° or 22° C.     

Screening of marine bacteria for fucoidanases

Twenty-five strains of epiphytic marine bacteria isolated from the brown algaeFucus evanescens andChorda filum and fifty-three bacteria isolated from the sea cucumberApostichopus japonicus were screened for fucoidanases using fucoidans prepared from the brown algaeF. evanescens, Laminaria cichorioides, andL japonica. Eighteen bacterial epiphytes and thirty-eight bacterial isolates from the sea cucumber were found to contain fucoidanases, which were able to hydrolyze either all of the fucoidans studied or some of them. Bacteria of the generaCytophaga andAlteromonas/Pseudoalteromonas exhibited the highest fucoidanase activities, which, however, did not exceed the activity of fucoidanases from the already known sources.     

The Seasonal Growth Dynamics of Fucus distichus subsp. evanescens (C. Agardh) H.T. Powell, 1957 (Phaeophyceae: Fucales) in the Avacha Bay (Southeastern Kamchatka)

Russian Journal of Marine Biology - The peculiarities of the linear growth and morphogenesis of the commercial brown alga Fucus distichus subsp. evanescens from the Avacha Bay (southeastern...     

Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds

Fucoidans and laminarans from Laminaria cichorioides, Laminaria japonica, Fucus evanescens, laminaran from Laminaria gurjanovae, other beta-D-glucans (translam, pustulan and zymosan) and lambda-carrageenan from Chondrus armatus were used to study the effect of water-soluble polysaccharides from seaweeds on the alternative pathway of complement (APC). beta-D-Glucans and fucoidans under study differed appreciably from each other by structural characteristics, and also by degree of purification. beta-D-glucans, on ability to bind complement, ranked in a line according to a degree of their purification. Highly purified beta-D-glucans under study did not reveal an ability to bind complement. The fucoidans were divided conventionally into three groups according to their action on APC. Highly sulfated alpha-L-fucan from L. cichorioides with the greatest activity toward APC and caused 50% inhibition of reaction of activation (RA) of APC in a concentration of 0.5-0.7 mg/ml. Opposite 50% of inhibition of lysis of erythrocytes by sulfated heterogeneous fucoidan from L. japonica was achieved with 20 mg/ml. All other fucoidans and lambda-carrageenan have activity at 6-10 mg/ml concentration. Decreasing the sulfate content from 36% up to 9% in sample fucoidans under study was not reflected practically in the 50% inhibition concentration. Apparently, the degree of sulfating of fucoidans did not influence their action on APC. But the positive influence of fucose in structure of polysaccharide was obvious.     

Gamete Release at Low Tide in Fucoid Algae: Maladaptive or Advantageous?

This review discusses three questions pertaining to gamete releaseby fucoid algae at low tide: 1) Are gametes viable and doesfertilization occur at low tide?, 2) How many gametes are releasedat low tide versus at high tide? and 3) Is gamete release atlow tide maladaptive or is it selectively advantageous? Gameterelease at low tide (LT) in fucoid algae is observed commonlyin monoecious species from the lower and mid-intertidal zones(e.g., Fucus distichus, F. evanescens, F. gardneri, Pelvetiacompressa); in dioecious species, intact antheridia (containingsperm) are commonly released at low tide (e.g., in Ascophyllumnodosum, F. vesiculosus). Fertilization at low tide can be determinedwith a calcofluor white assay and occurs in at least three species(F. distichus, F. gardneri, P. compressa). In general, fucoidalgae have high levels of fertilization success, but substantialmortality occurs during early embryogenesis in some intertidalzones due to physical stresses. The agarose bead assay is usefulto assess desiccation on an egg-sized scale. Constitutive dehydrin-likeproteins are present in sperm, eggs, and embryos. The releaseof gametes at low tide appears to be a consequence of a mechanismselected to permit gamete release under calm conditions in seawater;this mechanism is described. The timing of adhesion by zygotesat low and high tide is unknown. Such information and additionaldata on the relative proportions of zygotes produced by moneciousspecies at low tide versus high tide are required in order toassess effects of gamete release at low tide upon dispersaland population structure.     

Sulfated galactofucan from <Emphasis Type="Italic">Lobophora variegata</Emphasis>: Anticoagulant and anti-inflammatory properties

Sulfated polysaccharides (fucans and fucoidans) from brown algae show several biological activities, including anticoagulant and anti-inflammatory activities. We have extracted a sulfated heterofucan from the brown seaweed Lobophora variegata by proteolytic digestion, followed by acetone fractionation, molecular sieving, and ion-exchange chromatography. Chemical analyses and ¹³C-NMR and IR spectroscopy showed that this fucoidan is composed of fucose, galactose, and sulfate at molar ratios of 1:3:2. We compared the anticoagulant activity of L. variegata fucoidan with those of a commercial sulfated polysaccharide (also named fucoidan) from Fucus vesiculosus and heparin. The experimental inflammation models utilized in this work revealed that fucoidan from L. variegata inhibits leukocyte migration to the inflammation site. Ear swelling caused by croton oil was also inhibited when sulfated polysaccharides from F. vesiculosus and L. variegata were used. The precise mechanism of different action between homo-and heterofucans is not clear; nevertheless, the polysaccharides studied here may have therapeutic potential in inflammatory disorders. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 9, pp. 1265–1273.     

Content of alginic acid and fucoidan in fucus algae of the Barents sea

Seasonal changes in the content of alginic acid and fucoidan have been studied in four species of fucus algae from the Barents sea: Fucus vesiculosus, F. distichus, F. serratus, and Ascophyllum nodosum. These polysaccharides are accumulated in the biomass of the algae during summer and autumn. To ensure complete processing of fucus algae it is recommended that the raw material be harvested within this period.     

Physiological responses of intertidal marine brown algae to nitrogen deprivation and resupply of nitrate and ammonium

Intertidal macroalgae Fucus and Laminaria experience seasonally fluctuating inorganic N supply. This study examined the effects of long‐term N deprivation, recovery following N resupply, and effects of elevated ammonium and nitrate exposure on N acquisition in intertidal algae using manipulations of N supply in tank culture. Over 15 weeks of N deprivation, internal N and nitrate reductase activity (NRA) declined, but maximum quantum yield of PSII was unaffected in Fucus serratus and Fucus vesiculosus. Low NRA was maintained despite no external nitrate availability and depletion of internal pools, suggesting a constitutive NRA, insensitive to N supply. Nitrate resupplied to N‐starved thalli was rapidly taken up and internal nitrate pools and NRA increased. Exposure to elevated (50 μM) nitrate over 4 days stimulated nitrate uptake and NRA in Laminaria digitata and F. serratus. Exposure to elevated ammonium suppressed NRA in L. digitata but not in F. serratus. This novel insensitivity of NRA to ammonium in Fucus contrasts with regulation of NRA in other algae and higher plants. Ammonium suppression of NRA in L. digitata was not via inhibition of nitrate uptake and was independent of nitrate availability. L. digitata showed a higher capacity for internal nitrate storage when exposed to elevated ambient nitrate, but NRA was lower than in Fucus. All species maintained nitrate assimilation capacity in excess of nitrate uptake capacity. N uptake and storage strategies of these intertidal macroalgae are adaptive to life in fluctuating N supply, and distinct regulation of N metabolism in Fucus vs Laminaria may relate to position in the intertidal zone.     

EVOLUTION OF THE FUCACEAE (PHAEOPHYCEAE) INFERRED FROM nrDNA-ITS

Sequences of the internal transcribed spacer region (ITS-1, 5.8S, and ITS-2) of nuclear ribosomal DNA were obtained from 16 species representing all six genera of Fucaceae ( Ascophyllum, Fucus, Hesperophycus, Pelvetia, Pelvetiopsis, and Xiphophora ) plus one outgroup ( Hormosira ). Parsimony analysis indicated that the family Fucaceae is monophyletic and that the northern hemisphere taxa are highly divergent from the only southern hemisphere genus, Xiphophora. The genus Pelvetia is not monophyletic because the European P. canaliculata is more closely related to Fucus, Hesperophycus, and Pelvetiopsis than to other Pelvetia species. We establish Silvetia, gen. nov. and transfer the 3 Pacific species of Pelvetia to the new genus. Fucus is monophyletic and not ancestral in the Fucaceae. The ITS sequences identified two strongly supported lineages within Fucus, one with F. serratus sister to the clade containing F. gardneri, F. distichus, and F. evanescens and a second including F. vesiculosus, F. spiralis, F. ceranoides, and F. virsoides. The ITS was not useful for resolving relationships within each of these clusters and between populations of F. vesiculosus. Within-individual variation in ITS sequences is high in Fucus, a derived genus, compared to Ascophyllum, a more ancestral genus. Mapping of the two characters that form the basis of Powell's model for speciation in the Fucaceae showed that 1) number of eggs per oogonium has not followed a gradual reduction and that 2) monoecy/dioecy has changed several times during evolution of this family.     

CD226 mediates platelet and megakaryocytic cell adhesion to vascular endothelial cells

Platelet adhesion to vascular endothelial cells is a pathophysiologically relevant cell-to-cell interaction. However, the mechanisms underlying this cellular interaction are incompletely understood. In search of the ligand for CD226 adhesion molecule expressed on platelets, we found that human umbilical vein endothelial cells (HUVEC) express significant amount of putative CD226 ligand. We demonstrated that thrombin-activated, but not resting, platelets bind to intact HUVEC. Anti-CD226 monoclonal antibody specifically inhibited the binding, indicating that CD226 mediates the intercellular binding between thrombin-activated platelets and HUVEC. We also demonstrated that platelet activation with thrombin induces tyrosine phosphorylation of CD226 as well as CD226-mediated platelet adhesion. Moreover, experiments using mutant transfectants suggested that the tyrosine at residue 322 of CD226 plays an important role for its adhesive function. CD226 was also expressed on primary megakaryocytes and megakaryocytic cell lines. Anti-CD226 monoclonal antibody inhibited binding of megakaryocytic cell lines to HUVEC. Taken together, these results reveal a novel mechanism for adhesion of platelets and megakaryocytic cells to vascular endothelial cells.     

Antioxidant and pro-oxidant activities of the brown algae, Laminaria digitata, Himanthalia elongata, Fucus vesiculosus, Fucus serratus and Ascophyllum nodosum

The ability of Laminaria digitata, Himanthalia elongata, Fucus vesiculosus, Fucus serratus and Ascophyllum nodosum to scavenge peroxyl radicals was investigated by kinetic studies in a model system. The thermal initiated oxidation of methyl linoleate was performed at 60°C in heptanol, with or without antioxidants. When they reached 1% of the substrate, seaweed extracts exhibited antioxidant activities by extending the induction period, but they did not suppress the rate of oxygen uptake as did vitamin E. A synergistic effect occurred when both algal extracts (1.5 g L^-1) and vitamin E (0.4 mmol L^-1) were present, and the effectiveness of the combined antioxidants during the whole induction period was vitamin E effectiveness. The synergistic effect of L. digitata, however, was subject to seasonal variations: samples collected in summer were effective synergists, whereas samples collected in winter displayed a marked negative synergism. The phospholipid fractions of F. vesiculosus, F. serratus and A. nodosum, including pigments, accounted for only 6% of the total lipid fraction, and did not exhibit a large synergistic effect. The main phospholipid was not phosphatidyl ethanolamine as usually related, but phosphatidyl inositol. Fucoxanthin had some antioxidant activity per se under our experimental conditions, but did not act as a synergist of vitamin E. The most potent synergists were recognized as chlorophyll a and related compounds by the application of liquid-liquid partition and chromatography for the identification of active components. This revised version was published online in June 2006 with corrections to the Cover Date.