Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.
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A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper. (+info)
Structure and anticoagulant activity of sulfated fucans. Comparison between the regular, repetitive, and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae.
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Sulfated fucans are among the most widely studied of all the sulfated polysaccharides of non-mammalian origin that exhibit biological activities in mammalian systems. Examples of these polysaccharides extracted from echinoderms have simple structures, composed of oligosaccharide repeating units within which the residues differ by specific patterns of sulfation among different species. In contrast the algal fucans may have some regular repeating structure but are clearly more heterogeneous when compared with the echinoderm fucans. The structures of the sulfated fucans from brown algae also vary from species to species. We compared the anticoagulant activity of the regular and repetitive fucans from echinoderms with that of the more heterogeneous fucans from three species of brown algae. Our results indicate that different structural features determine not only the anticoagulant potency of the sulfated fucans but also the mechanism by which they exert this activity. Thus, the branched fucans from brown algae are direct inhibitors of thrombin, whereas the linear fucans from echinoderms require the presence of antithrombin or heparin cofactor II for inhibition of thrombin, as reported for mammalian glycosaminoglycans. The linear sulfated fucans from echinoderms have an anticoagulant action resembling that of mammalian dermatan sulfate and a modest action through antithrombin. A single difference of one sulfate ester per tetrasaccharide repeating unit modifies the anticoagulant activity of the polysaccharide markedly. Possibly the spatial arrangements of sulfate esters in the repeating tetrasaccharide unit of the echinoderm fucan mimics the site in dermatan sulfate with high affinity for heparin cofactor II. (+info)
Influence of centriole behavior on the first spindle formation in zygotes of the brown alga Fucus distichus (Fucales, Phaeophyceae).
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The influence of centrioles, derived from the sperm flagellar basal bodies, and the centrosomal material (MTOCs) on spindle formation in the brown alga Fucus distichus (oogamous) was studied by immunofluorescence microscopy using anti-centrin and anti-beta-tubulin antibodies. In contrast to a bipolar spindle, which is formed after normal fertilization, a multipolar spindle was formed in polyspermic zygote. The number of mitotic poles in polyspermic zygotes was double the number of sperm involved in fertilization. As an anti-centrin staining spot (centrioles) was located at these poles, the multipolar spindles in polyspermic zygotes were produced by the supplementary centrioles. When anucleate egg fragments were fertilized, chromosome condensation and mitosis did not occur in the sperm nucleus. Two anti-centrin staining spots could be detected, microtubules (MTs) radiated from nearby, but the mitotic spindle was never produced. When a single sperm fertilized multinucleate eggs (polygyny), abnormal spindles were also observed. In addition to two mitotic poles containing anti-centrin staining spots, extra mitotic poles without anti-centrin staining spots were also formed, and as a result multipolar spindles were formed. When karyogamy was blocked with colchicine, it became clear that the egg nucleus proceeded independently into mitosis accompanying chromosome condensation. A monoastral spindle could be frequently observed, and in rare cases a barrel-shaped spindle was formed. However, when a sperm nucleus was located near an egg nucleus, the two anti-centrin staining spots shifted to the egg nucleus from the sperm nucleus. In this case, a normal spindle was formed, the egg chromosomes arranged at the equator, and the associated MTs elongated from one pole of the egg spindle toward the sperm chromosomes which were scattered. From these results, it became clear that paternal centrioles derived from the sperm have a crucial role in spindle formation in the brown algae, such as they do during animal fertilization. However, paternal centrioles were not adequate for the functional centrosome during spindle formation. We speculated that centrosomal materials from the egg cytoplasm aggregate around the sperm centrioles and are needed for centrosomal activation. (+info)
Isolation and characterization of a Ca2+ -binding polysaccharide associated with coccoliths of Emiliania huxleyi (Lohmann) Kamptner.
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C-occolithophoridae, a group of mostly unicellular algae, possess a cell wall containing calcified plates, called coccoliths. The coccoliths from the species Emilania huxleyi (Lohmann) Kamptner contain a water-soluble acid polysaccharide. In this paper we describe the isolation and some characteristic properties of the polysaccharide, in particular its Ca2+ -binding capacity. A large-scale cultivation of the Coccolithophoridae was worked out and a new procedure for isolating coccoliths was developed. The polysaccharide obtained from the coccoliths contained two types of monobasic acid groups in a total amount of 1.8 mumol/mg polysaccharide. One type consisted of weakly acid groups which were identified as uronic acids. The nature of the stronger acid groups remains to be established. The ratio between the respective groups was 1:0.8. Studies with 45Ca2+ demonstrated that the isolated polysaccharide is capable of binding Ca2+. Equilibrium dialysis revealed that the maximum amount of Ca2+ which can be bound in 0.92 +/- 0.05 mumol/mg polysaccharide. Flow-rate dialysis experiments strongly suggested the presence of two classes of Ca2+ -binding sites differing in affinity for Ca2+. High-affinity sites (dissociation constant Kd for Ca2+ :2.2 +/- 1.0 X 10(-5) M) were found to be present in amounts (0.38 +/- 0.04 mumol/mg polysaccharide) approximately equivalent to the strongly acid monovalent groups mentioned above (0.8 mumol/mg polysaccharide). Low-affinity sites (Kd for Ca2+: -11 +/- 39 X 10(-5) M) were estimated at 0.74 +/- 0.11 mumol/mg polysaccharide. Although this figure could be determined less accurately, it is suggested that the uronic acids (1.0 mumol/mg polysaccharide) are identical to the low-affinity sites. Preferential binding of Ca2+ occurred in a 100-fold excess of Na+ and Mg2+ as was shown by gel filtration. A 100-fold excess of Sr2+ inhibited Ca2+ binding to a great extent while no Ca2+ was bound in the presence of an equimolar amount of La3+. The dissociation constants of the high-affinity sites for Na+, Mg2+, Sr2+ and La3+ (in the presence of Ca2+) were determined with the flow-rate dialysis technique. They confirm the order of binding preference found with gel filtration. A polysaccharide with similar properties could be isolated from subfossil coccoliths of E. hyxleyi (about 1000 years old). The possible role of the polysaccharide as a heterogeneous matrix in coccolith formation is discussed. (+info)
Persistent virus integration into the genome of its algal host, Ectocarpus siliculosus (Phaeophyceae).
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The brown alga Ectocarpus siliculosus frequently carries an endogenous virus, E. siliculosus virus (EsV-1), the genome of which is a circular, double-stranded DNA molecule of about 320 kbp. After infection, which occurs in the unicellular spores or gametes, the virus is present latently in all somatic cells of the host. Virus multiplication is restricted to cells of the reproductive organs. It has been an open question whether the latent viral DNA occurs as a free episome or becomes integrated into the host genome. PCR studies showed that viral DNA co-migrates with high molecular mass DNA in pulsed-field gel electrophoresis, which confirms that latent viral DNA is integrated into the host genome. (+info)
Description of Cellulophaga baltica gen. nov., sp. nov. and Cellulophaga fucicola gen. nov., sp. nov. and reclassification of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb. nov.
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Phenotypic data indicate that gliding, yellow/orange-pigmented, agar-digesting bacterial strains were members of the Cytophaga-Flavobacterium-Bacteroides (CFB) group. The strains were isolated from the surface of the marine benthic macroalga Fucus serratus L. and the surrounding seawater at three localities in Danish waters. The bacteria were Gram-negative, flexirubin-negative, aerobic, catalase-positive and oxidase-negative and were psychrophilic and halophilic. All strains utilized D-fructose, L-fucose and alpha-ketobutyric acid and degraded alginic acid, carrageenan, starch and autoclaved yeast cells. Amplification with primers specific for repetitive extragenic palindromic elements by PCR divided the strains of this study into two groups. Both groups showed unique PCR amplification patterns compared to reference strains of the CFB group. Phylogenetic analysis of 16S rDNA sequences showed association of these organisms and [Cytophaga] lytica at the genus level. Hybridization of total chromosomal DNA revealed that the new strains and [Cytophaga] lytica ATCC 23178T were clearly distinct from each other and other previously described species of the CFB group. A new genus is described, Cellulophaga gen. nov. comprising two new species, Cellulophaga baltica gen. nov., sp. nov. (NN015840T = LMG 18535T) and Cellulophaga fucicola gen. nov., sp. nov. (NN015860T = LMG 18536T), as well as the emendation of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb. nov. (+info)
Bromine K-edge EXAFS studies of bromide binding to bromoperoxidase from Ascophyllum nodosum.
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Bromine K-edge EXAFS studies have been carried out for bromide/peroxidase samples in Tris buffer at pH 8. The results are compared with those of aqueous (Tris-buffered) bromide and vanadium model compounds containing Br-V, Br-C(aliphatic) and Br-C(aromatic) bonds. It is found that bromide does not coordinate to the vanadium centre. Rather, bromine binds covalently to carbon. A possible candidate is active site serine. (+info)
Inhibition of the establishment of zygotic polarity by protein tyrosine kinase inhibitors leads to an alteration of embryo pattern in Fucus.
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Fucoid algae, including the genus Fucus and Pelvetia, are recognized as model systems to study early embryogenesis in plants. In particular the zygotes of these fucoid algae are highly suitable experimental systems for investigating the establishment of polarity and its requirement for later embryogenesis. However, the transduction pathways involved in the initiation of polarization are still poorly understood, and the link between the early polarization processes and embryo long-term patterning has never been experimentally demonstrated. We, therefore, have investigated the putative role of protein phosphorylation in the regulation of early embryogenesis, using a combined pharmacological and biochemical approach. Among the various protein kinase inhibitors tested, a subset of well-known PTK inhibitors, including genistein, prevented germination but had no effect on growth of germinated zygotes and embryos. Inhibition of germination appeared to be a direct consequence of prevention of polarization since genistein and other PTK inhibitors specifically inhibited axis formation in a light-independent manner. Genistein inhibited cellular events associated with polarization such as polarized secretion of cell wall sulfated compounds. Anchorage of F-actin at the rhizoid pole was also inhibited and F-actin redistributed in response to a new light vector. Zygotes inhibited in the polarization process over the period of axis formation recovered from the treatment and displayed differentiated cellular structures after a few days. However, they exhibited a deeply disorganized pattern, suggesting that the early polarization process is essential for normal patterning of the embryo. Western blot analysis of protein phosphorylation showed that the patterns of protein phosphorylation changed during development and were disturbed by treatments with genistein. This drug also inhibited in vitro autophosphorylation. The nature of the genistein-sensitive kinases required for polarization and long-term patterning is discussed in light of these data. (+info)