Agglutinins from marine macroalgae of the southeastern United States

Protein extracts from 22 species of marine macroalgae from Florida and North Carolina were compared for their abilities to agglutinate sheep and rabbit erythrocytes. Protein extracts from 21 algal species agglutinated rabbit erythrocytes compared to 19 for sheep erythrocytes. However, agglutination by brown algal extracts was variable. The agglutination produced by protein extracts from Dictyota dichotoma could be blocked by addition of polyvinylpyrrolidone. Protein extracts from North Carolina macroalgae were also tested against five bacterial species. Three of these agglutinated bacterial cells. Ulva curvata and Bryopsis plumosa agglutinated all five species. Protein extracts from five species of Florida algae were tested for their effects on mitogenesis in mouse splenocytes and human lymphocytes. Gracilaria tikvahiae HBOI Strain G-5, Ulva rigida and Gracilaria verrucosa HBOI Strain G-16S stimulated mitogenesis in mouse splenocytes, while Gracilaria tikvahiae HBOI Strain G-16stimulated mitogenesis in human lymphocytes.     

Haemagglutinating and antibiotic activities of freshwater microalgae

We analysed the haemagglutinating activity of algal extracts from 44 species of freshwater microalgae against native and trypsin/papain-treated cow, pig, sheep, and human A-, B-, and O-type erythrocytes. Algal extracts obtained with aqueous ethanol exhibited higher haemagglutinating activity than those obtained with aqueous acetone. Most of the algal extracts agglutinated at least one of the erythrocyte types analysed. Human erythrocytes were the most sensitive of the cell types analysed. In the other species, the sensitivity of algal haemagglutinating activity for erythrocytes was pig > sheep > cow. Pre-treating erythrocytes with trypsin and papain improved the detection of most algal agglutinins and increased the haemagglutination titre; pre-treatment with papain was most effective for pig erythrocytes. Algal extracts stored at –20 °C for 4 months lost their haemagglutinating activity. Algal extracts also exhibited strong antibiotic activity against food pathogenic bacteria, especially against Bacillus. Our numerical taxonomy data showed that these microalgae might be grouped into several clusters according to their haemagglutinating activity. The detection of haemagglutinating activity may provide an efficient biochemical or physiological character to classify and differentiate microalgae. Our results suggest that freshwater microalgae might provide a potent source of haemagglutinins and antibacterial compounds for biochemical and medical studies and applications.     

Agglutination of human and animal erythrocytes in marine unicellular algae

Ethanol extracts of 12 marine unicellular algae were assayed for agglutinating activity against native and enzyme-treated human and animal erythrocytes. All of the algae assayed agglutinated at least one group of normal erythrocytes from humans. Notably, all algal extracts agglutinated erythrocytes of hemophilia patients arising from coagulation disorders. Meanwhile, all algae had a strong reaction with monkey erythrocytes, but to a lesser extent or not at all with sheep erythrocytes. Both trypsin and pronase improved the detection of most algal agglutinins and caused a drastic increase in hemagglutinating activity after treatment for 2 h or more. However, hemagglutinating activity decreased or disappeared completely when two extracts of different algal species were combined. Journal of Industrial Microbiology & Biotechnology (2000) 24, 262–266. Received 24 September 1999/ Accepted in revised form 06 January 2000     

A comparative study of animal erythrocyte agglutinins from marine algae

1. Fifteen marine algal species were analyzed for agglutinins to rabbit, sheep and human A, B and O blood group erythrocytes. 2. Protein extracts from all marine algae agglutinated rabbit erythrocytes, whereas twelve and five extracts agglutinated sheep and human erythrocytes, respectively. 3. The highest agglutination titers were consistently observed with rabbit erythrocytes. 4. Dictyota dichotoma strongly agglutinated human B blood group erythrocytes relative to A and O group erythrocytes. 5. Agglutination titer of rabbit erythrocytes by six algal extracts was not inhibited by mono- or polysaccharides, yet was reduced by glycoproteins.     

Two coexisting tank bromeliads host distinct algal communities on a tropical inselberg

The tank bromeliads Aechmea aquilega (Salisb.) and Catopsis berteroniana (Schultes f.) coexist on a sun‐exposed Neotropical inselberg in French Guiana, where they permit conspicuous freshwater pools to form that differ in size, complexity and detritus content. We sampled the algal communities (both eukaryotic and cyanobacterial taxa, including colourless forms) inhabiting either A. aquilega (n = 31) or C. berteroniana (n = 30) and examined differences in community composition and biomass patterns in relation to several biotic and abiotic variables. Chlorella sp. and Bumilleriopsis sp. were the most common taxa and dominated the algal biomass in A. aquilega and C. berteroniana, respectively. Using a redundancy analysis, we found that water volume, habitat complexity and the density of phagotrophic protozoa and collector‐gatherer invertebrates were the main factors explaining the distribution of the algal taxa among the samples. Hierarchical clustering procedures based on abundance and presence/absence data clearly segregated the samples according to bromeliad species, revealing that the algal communities in the smaller bromeliad species were not a subset of the communities found in the larger bromeliad species. We conclude that, even though two coexisting tank bromeliad populations create adjacent aquatic habitats, each population hosts a distinct algal community. Hence, bromeliad diversity is thought to promote the local diversity of freshwater algae in the Neotropics.     

A new screening for hemagglutinins from Vietnamese marine macroalgae

Aqueous extracts from 42 species of Vietnamese marine macroalgae, including 17 Chlorophyta, 22 Rhodophyta, and three Phaeophyta species, were examined for hemagglutination activity using native and enzyme-treated different animal and human erythrocytes. All extracts agglutinated at least one type of erythrocytes tested. Strong activity was detected in extracts from four Chlorophyta (Caulerpa serulata var. boryana, Caulerpa sertularioides f. longipes, Halimeda velasquezii, and Halimeda discoidea) and two Rhodophyta species (Gelidiella acerosa and Titanophora pulchra) with enzyme-treated rabbit and horse erythrocytes. The hemagglutinins of some active species were examined for sugar-binding specificity, pH, temperature stability, and divalent cation independency using ammonium sulfate precipitates prepared from their extracts. In a hemagglutination–inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides. The activity of the hemagglutinins was inhibited by some glycoproteins tested. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, suggesting the presence of lectins specific for complex N-glycans, high mannose N-glycans or O-glycans. On the other hand, the activities of almost all algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations, except Gelidiopsis scoparia hemagglutinin, its activity was dependent on the presence of divalent cations. These results suggest that Vietnamese marine macroalgae may be good sources of useful lectins for many biological applications.     

Screening microalgae for some potentially useful agricultural and pharmaceutical secondary metabolites

Nearly two hundred microalgal strains (174 Chlorophyta and 23 Cyanobacteria) were screened against some bacteria, filamentous fungi and yeasts using a disc-diffusion type bioassay. From this initial screening, 10 Chlorophyta strains from three genera (Desmococcus, Chlorella and Scenedesmus) were selected because of their high antimicrobial activity. These 10 strains were partially purified and tested using MIC antimicrobial and microtiter IC₅₀ anticancer assays. These preselected algal strains showed a high incidence of antibacterial activity against both Gram-positive (9 out of 10 species) and Gram-negative (7 out of 10 species) bacteria. The extracts were also effective against some tumour cell lines.     

Electron microscopy of human erythrocytes agglutinated by lectin from Codium fragile ssp. tomentosoides and pseudo-haemagglutinin from Ascophyllum nodosum

Human erythrocytes were exposed to extracts of Codium fragile ssp. tomentosoides, Ascophyllum nodosum, Dolichos biflorus seeds and a solution of purified polyphenols from Ascophyllum nodosum. In each case the erythrocytes appeared to agglutinate. Agglutinates were examined by scanning electron microscopy. Photomicrographs of the erythrocytes exposed to lectin-containing extracts of C. fragile and D. biflorus showed the typical appearance of agglutinated cells. Photomicrographs of the erythrocytes exposed to the crude extract and purified polyphenols from A. nodosum were similar to each other, but quite different in appearance to erythrocytes agglutinated by the lectins from C. fragile and D. biflorus. This evidence adds further support to the contention that lectin presence recorded in brown algal extracts is largely due to ‘pseudoagglutination’ produced by polyphenols contained in these extracts.     

Characterization of a Monoclonal Antibody Specific for Lipoteichoic Acid from Various Gram-Positive Bacteria

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.     

Bacteroides gingivalis,Bacteroides asaccharolyticus,andBacteroides melaninogenicus subspecies: Cell surface morphology and adherence to erythrocytes and human buccal epithelial cells

All study strains ofBacteroides gingivalis, B. asaccharolyticus, andB. melaninogenicus subspecies possessed numerous pilus-like fibers and capsule-like outer surface structures. The capsular morphology varied between the different species and subspecies.B. gingivalis strongly agglutinated 16 erythrocyte species studied.B. asaccharolyticus showed variable and weak agglutination of only a few erythrocyte species.B. melaninogenicus subsp.intermedius strains strongly agglutinated rabbit erythrocytes and exhibited variable, often weak agglutination of 8 other erythrocyte species. Preparations of capsular polysaccharide or lipopolysaccharide fromB. gingivalis failed to agglutinate human erythrocytes, while pili preparations from the same organisms possessed marked hemagglutinating activity.B. gingivalis cells adhered in high numbers to human buccal epithelial cells, whereas strains ofB. asaccharolyticus failed to show measurable adherence. Oral strains ofB. melaninogenicus subsp.intermedius feebly adhered to the buccal epithelial cells. Pretreatment ofB. gingivalis cells with serum or saliva prevented the adherence to epithelial cells. Our findings suggest that cell surfaces with distinct properties exist on the various black-pigmentedBacteroides species and subspecies and this may accout for markedly differing ability of these organisms to attach to mammalian cells.     

The use of xenoantigenic antisera for the identification of tilapiine species: comparative laboratory and field studies

Rapid and reliable identification of tilapiine taxa and strains is essential for selective breeding purposes and the conservation of natural genetic resources. There is evidence that antisera‐mediated erythrocyte agglutination assays can fit these requirements. We evaluated the applicability of agglutination tests by studying the capacity of species characteristic antisera to recognize erythrocytes from individuals of 10 natural Ghanaian populations of Oreochromis niloticus and Sarotherodon melanotheron. The vast majority of the 218 tested individuals could be identified based on antisera‐mediated erythrocyte recognition. Controls indicated the specificity of these reactions. Still, erythrocytes from 16% of all tested specimens did not respond to any antiserum (zero responders), indicating the possible existence of blood group properties in tilapias. We discuss the specificity of the antisera, the relevance of zero responses and the applicability of these tests in aquaculture and field studies.     

A new survey of Brazilian marine algae for agglutinins

Aqueous protein extracts from 30 Brazilian marine algae were examined for haemagglutinating activity using native and enzyme-treated rabbit, chicken, sheep and human erythrocytes. Most extracts agglutinated at least one of the blood cells used. Sheep and rabbit erythrocytes were more suitable for detection of the agglutinating activity. The minimum protein concentration necessary to produce positive agglutination was usually lower with enzyme-treated erythrocytes than native ones. The five algal protein extracts showing the greatest haemagglutination titre were tested for sugar-binding specificity. Only the activity present in the green alga Cauler pacupressoides was inhibited by simple sugars and not by the glycoproteins tested. The activity of the other four extracts was inhibited by at least one of the glycoproteins utilised. This revised version was published online in August 2006 with corrections to the Cover Date.     

Preparation and properties of antisera to glycolipid of guinea pig erythrocyte membrane

Antibodies against a glycolipid of guinea pig erythrocyte membranes were prepared in rabbits by immunization with guinea pig erythrocyte stroma or the purified glycolipid, gangliotriaosylceramide. The antibodies agglutinated guinea pig erythrocytes. The specificity of antibodies could be revealed by several immunochemical methods, including inhibition of hemagglutination, immunodiffusion, agglutination of liposomes, and complement fixation. The antibodies were specific for gangliotriaosylceramide.     

Cyanide formation in preparations from Chlorella and New Zealand spinach leaves: Effect of added amino acids

Summary As part of an effort to identify the natural precursor(s) of HCN in the alga Chlorella vulgaris Beijerinck, and in leaves of New Zealand spinach (Tetragonia expansa, Murr.), HCN release was measured after addition of various amino acids to illuminated algal extracts and grana preparations. Histidine is particularly effective as an HCN precursor, both with Chlorella extracts and leaf grana. With the algal extracts, d-histidine is about ten times more effective than l-histidine and histamine, whereas the two isomers (and histamine) are about equally effective with leaf grana. In the presence of leaf grana plus added Mn²⁺ and peroxidase, l-tyrosine and l-cysteine like-wise cause HCN formation; but these amino acids cause little or no HCN formation in the presence of Chlorella extracts. A stimulation of HCN production by l-histidine was observed with intact Chlorella cells. Because of the limitations of the assay method, the possibility can not be excluded that other substances than histidine may also lead to HCN generation in Chlorella vulgaris, but the results show that histidine has an important role in HCN generation by this species.Abbreviation POD peroxidase     

Deep genomic analysis of the Chlorella sorokiniana SAG 211-8k chloroplast

The chloroplast genome contains information that is applicable in many scientific fields, such as plant systematics, phylogenetic reconstruction and biotechnology, because its features are highly conserved among species. To date, several complete green algal chloroplast genomes have been sequenced and assembled. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Chlorella sorokiniana SAG 211-8k is reported and compared for the first time to the chloroplast genomes of 10 Chlorellaceae. The recently updated Chlorella sorokiniana cpDNA sequence, assembled as a circular map of 109?811 bp, encodes 113 genes. Similar to other Chlorella strains, this chloroplast genome does not show a quadripartite structure and lacks the large rRNA operon-encoding Inverted Repeat (IR). The Chlorella sorokiniana plastid encodes the tRNA(Ile)-lysidine synthetase (tilS), which is responsible for modifying the CAU anticodon of a unique tRNA. Gene ordering and clustering highlight the close relationships among Chlorella clade members and the preservation of crucial gene clusters in photosynthetic strains. The features of Chlorella sorokiniana presented here reinforce the monophyletic character of Chlorellaceae and provide important information that sheds light on chloroplast genome evolution among species of Chlorella.     

LECTIN-LIKE COMPOUNDS AND LECTIN RECEPTORS IN MARINE MICROALGAE: HEMAGGLUTINATION AND REACTIVITY WITH PURIFIED LECTINS1

We detected lectin-like compounds and lectin receptors in microalgae by hemagglutination, competitive inhibition with sugars, and reactivity with lectins isolated from other sources. Cell extracts from eight species of Dinophyceae and from one species each of Raphidophyceae and Bacillariophyceae exhibited hemagglutination toward trypsinized rabbit erythrocytes. In addition, the culture media of the dinoflagellate Alexandrium cohorticula and the raphidophyte Chattonella antiqua displayed similar hemagglutination. These activities were not inhibited by any monosaccharides or oligosaccharides tested but were inhibited by some specific glycoproteins. This suggests that the active factors were lectin-like compounds. Upon exposing intact, healthy cells of 12 species of Dinophyceae and one species each of Raphidophyceae, Cryptophyceae, Bacillariophyceae, and Chlorophyceae to lectins isolated from either macroalgae or terrestrial plants, most species were adversely affected. The negative effects included one or more of the following: impaired motility, disappearance of motility, agglutination, abnormal morphology, and cell rupture or lysis. Some species, even after freezing, thawing, and washing with saline solution, still agglutinated with macroalgal or terrestrial plant lectins. This study suggests that lectins and carbohydrate-containing lectin receptors may commonly occur on the cell surfaces of various species of microalgae.     

New species Micractinium kostikovii (Chlorellaceae,Trebouxiophyceae) from Russia

Correctly identifying species of Chlorella-like microalgae is difficult because of their morphological simplicity and high phenotypic plasticity. The use of molecular tools has revolutionized research on algal diversity, enabling such advances as the discovery of numerous new taxa. This article presents the results of a study of strains that are newly isolated from a freshwater Lake Prudovikov (Samara region, Russian Federation). These strains had the typical Chlorella morphology, exhibiting spherical cells and a cup-shaped parietal chloroplast. The chloroplast contained a single pyrenoid enveloped by starch grains. However, 18S–ITS1–5.8S–ITS2 sequence analyses indicated that the studied strains are strongly allied with the well-supported genus Micractinium. Based on the results of a comparative analysis of the morphological, molecular and environmental characteristics of the studied strains (employing a polyphasic approach), we propose that they are new species of Micractinium: Micractinium kostikovii sp. nov.     

Haemagglutinin activity in Acrididae (grasshopper) haemolymph

The haemolymph of Acrididae causes haemagglutination of human and animal erythrocytes. Thirteen of seventeen species tested had detectable activity and gave agglutination titres in the range 2–64, Melanoplus bivittatus, and M. sanguinipes showed greatest activity. Haemagglutinin activity is continuously present in male and female insects from 4th instar and throughout adulthood. Females contain slightly more activity than do males. M. sanguinipes haemolymph agglutinates rabbit, calf, human (all ABO types) guinea pig, mouse, chicken, cat, pig and sheep erythrocytes. Rabbit red cells are agglutinated most strongly and sheep and chicken cells least. M. sanguinipes haemolymph also agglutinates the protozoan Nosema locustae, a natural grasshopper pathogen. Preabsorption of haemolymph with different erythrocyte types selectively removes haemagglutinin activity suggesting the presence of multiple or heteroagglutinins. M. sanguinipes haemagglutinin is inhibited by glycoproteins, simple carbohydrates and carbohydrate derivatives. The inhibitory pattern is complex and among the sugars tested only mannose and derivatives of mannose are exclusively non-inhibitory. Haemolymph haemagglutinin activity is destroyed by heat and EDTA. It is totally precipitated by dialysis against water and may be partially recovered in phosphate or Tris buffer. Activity is stable in frozen haemolymph.     

梨形环棱螺凝集素的初步研究

通过Sepharose 4B-甲状腺球蛋白亲和层析,从梨形环棱螺Bellamya purificata体内分离到的一种凝集素,不连续PAGE显示其为单一的蛋白质谱带.它能凝集兔、猪、鸭等动物的红细胞,但不能凝集人的A、B、O及AB型血的红细胞和固定后的兔红细胞.其凝集活力可被1.0mol/L的乳糖、半乳糖和60g/L的甲状腺球蛋白抑制,但不能被碱性硼酸缓冲液抑制.对温度变化敏感,有较宽的最适pH范围.     

Generic concept in Chlorella‐related coccoid green algae (Chlorophyta,Trebouxiophyceae)

Using a combined set of sequences of SSU and ITS regions of nuclear‐encoded ribosomal DNA, the concept of the experimental algal genus Chlorella was evaluated. Conventionally in the genus Chlorella, only coccoid, solitary algae with spherical morphology that do not possess any mucilaginous envelope were included. All Chlorella species reproduce asexually by autospores. However, phylogenetic analyses showed that within the clade of ‘true’Chlorella species (Chlorella vulgaris, C. lobophora, and C. sorokiniana), taxa with a mucilaginous envelope and colonial lifeform have also evolved. These algae, formerly designated as Dictyosphaerium, are considered as members of the genus Chlorella. In close relationship to Chlorella, five different genera were supported by the phylogenetic analyses: Micractinium (spherical cells, colonial, with bristles), Didymogenes (ellipsoidal cells, two‐celled coenobia, with or without two spines per cell), Actinastrum (ellipsoidal cells within star‐shaped coenobia), Meyerella (spherical cells, solitary, without pyrenoids), and Hegewaldia (spherical cells, colonial, with or without bristles, oogamous propagation). Based on the secondary structures of SSU and ITS rDNA sequences, molecular signatures are provided for each genus of the Chlorella clade.