Ptilota Filicina Research Articles

Feeding of the short-spined sea urchin Strongylocentrotus intermedius on macroalgae and benthic animals

To examine the feeding ecology of the sea urchin Strongylocentrotus intermedius, two types of macroalgal diet (kelp Saccharina longissima and red alga Ptilota filicina) and four types of benthic animal diet [barnacle Balanus glandula, limpet Lottia cassis, free-living Perinereis aibuhitensis and P. aibuhitensis attached to a stainless steel wire (wired polychaete)], were separately or simultaneously provided for urchins of 5- to 60-mm test diameter (TD). Sea urchins of even 3-mm TD could graze the kelp, and began to graze the red alga, the wired polychaete and limpet by 5-mm TD, the barnacle by 8-mm TD and the free-living polychaete by 15-mm TD, respectively. These results suggest that the variety of macroalgae and benthic animals which the urchin can graze increases with the ontogeny of their feeding organs, motility and ability to catch animals. Both juvenile and adult sea urchins preferred to eat the polychaete as well as the kelp fronds. Feed conversion efficiency and daily growth rate of the small sea urchins fed benthic animals like the polychaete were higher than those fed macroalgae. The sea urchins are likely to grow better when feeding on animals than on macroalgae, even in habitats where they can access adequate quantities of kelp.

The interspecific relationships of seaweeds and their role in the formation of communities of Ahnfeltia tobuchiensis (Kanno et Matsubara, 1932) Makienko, 1970 (Rhodophyta)

The character and intensity of interspecific relationships between common species of an Ahnfeltia bed community (Ahnfeltia tobuchiensis, Ahnfeltiopsis flabelliformis, Chondrus armatus, and Ptilota filicina) were investigated in relation to light levels and concentrations of dissolved inorganic nitrogen and phosphorus compounds. Growth and photosynthetic rates of seaweeds were measured in mono and co-cultures, as well as in a medium containing the metabolites of attendant species. Under low light conditions, A. tobuchiensis showed the highest competitive ability among the species we studied. The experimental results indicated both negative (exploitative competition, allelopathic inhibition of production processes of a competitor) and positive (stimulation of growth and photosynthesis of accompanying species) relationships between seaweeds. Increased nutrient concentrations and light levels resulted in a substantial reduction in the intensity of interspecific relationships. The dynamic system of positive and negative relationships may regulate the A. tobuchiensis community structure; the intensity of interspecific relationships depends on the availability of resources such as light and nutrients.

Spatial distribution of algae in the Ahnfeltia tobuchiensis bed in Amursky Bay (Sea of Japan)

Horizontal and vertical distribution of algae in Ahnfeltia tobuchiensis beds was studied in the area of Mt. Stolovaya in Amursky Bay, Sea of Japan. Thirty-four species of macroalgae (2 species of Chlorophyta, 26 Rhodophyta, and 6 Ochrophyta) were found in the area of study. Two fields of the unattached alga Ahnfeltia were located opposite Mt. Stolovaya; they differed in area, macrophyte stock, number and biomass of attendant species, hydrochemical and light conditions. A monodominant Ahnfeltia tobuchiensis community formed in the southern field and a bidominant community of Ahnfeltia tobuchiensis + Ahnfeltiopsis flabelliformis developed in the northern field. It is suggested that the horizontal distribution of common macrophyte species in the Ahnfeltia beds at Mt. Stolovaya is conditioned by the amount of photosynthetically active radiation (PAR) reaching the surface of the algal bed. Differences in tolerance of Ahnfeltia and attendant species to shadowing and in light conditions across the alga stratum define the vertical distribution of algae within the bed. Chondrus armatus and Ptilota filicina were found mainly in the upper layer of the algal bed. A. tobuchiensis, A. flabelliformis, Chaetomorpha linum, and Coccotylus orientalis extended across the entire thickness of the bed.

Biochemistry of PUFA Double Bond Isomerases Producing Conjugated Linoleic Acid

The biotransformation of linoleic acid (LA) into conjugated linoleic acid (CLA) by microorganisms is a potentially useful industrial process. In most cases, however, the identities of proteins involved and the details of enzymatic activity regulation are far from clear. Here we summarize available data on the reaction mechanisms of CLA-producing enzymes characterized until now, from Butyrivibrio fibrisolvens, Lactobacillus acidophilus, Ptilota filicina, and Propionibacterium acnes. A general feature of enzymatic LA isomerization is the protein-assisted abstraction of an aliphatic hydrogen atom from position C-11, while the role of flavin as cofactor for the double bond activation in CLA-producing enzymes is also discussed with regard to the recently published three-dimensional structure of an isomerase from P. acnes. Combined data from structural studies, isotopic labeling experiments, and sequence comparison suggest that at least two different prototypical active site geometries occur among polyunsaturated fatty acid (PUFA) double bond isomerases.

High Pressure Freezing (HPF)을 이용한 조류 Ptilota filicina의 미세구조 관찰:HPF 고정법과 화학 고정법의 비교

In preparation of the biological samples for electron microscopy, the chemical fixation by glutaraldehyde, paraformaldehyde, and OsO4 has been generally used for a long time. However, the chemical fixation method has some problems: the infiltration time is a little bit long and the ultrastructure of cell or tissue transforms before complete fixation of sample. So, recently, cryo-fixation is considered more often in biomedical field. In this study, we compared High Pressure Freezing (HPF) method with chemical fixation method using a algal sample (Ptilota filicina J. Agardh), which was difficult to fix using chemical fixation method. In chloroplast, the ultrastructure of thylakoid lamella and phycobilisome can not show clearly by chemical fixation. In this study we could observe the ultrastructure of thylakoid lamella and phycobilisome of chloroplast very clearly using HPF fixation. An improved images of ultrastructures of nucleus, mitochondrion and floridean starch could obtain. These results suggest that HPF method is very useful method in algal specimen for electron microscopy.

Synthesis of the conjugated trienes 5E,7E,9E,14Z,17Z‐eicosapentaenoic acid and 5Z,7E,9E,14Z,17Z‐eicosapentaenoic acid, and their induction of apoptosis in DLD‐1 colorectal adenocarcinoma cells

During the course of our recent study on the anti-tumor effect of conjugated eicosapentaenoic acids (CEPA), we found that acid mixtures prepared by treating EPA with KOH in ethylene glycol induced potent apoptotic cell death in human tumor cells via membrane phospholipid peroxidation. Interestingly, the KOH-treated CEPA mixtures were more cytotoxic than EPA and CLA and had no effect on normal human fibroblast cells. To identify the specific cytotoxic FA in the CEPA mixture, we synthesized possible candidates for the active species. Here, we report the synthesis of (5E,7E,9E, 14Z, 17Z)-5,7,9,14,1 7-eicosapentaenoic acid (E-CEPA) and its 5-(Z) isomer (Z-CEPA), both of which are conjugated trienes that exist naturally in red algae (Ptilota filicina J. Agardh). E-CEPA and Z-CEPA were synthesized from methyl 5-oxopentanoate in six steps, using three types of Wittig reactions as the key steps. Next, we examined the cytotoxicity of E-CEPA and Z-CEPA in human tumor cells and confirmed their bioactivity. Both E-CEPA and Z-CEPA had a strong cytotoxic reaction in tumor cells, and this effect occurred through induction of apoptosis.

Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA

The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS–PAGE and silver staining). By sedimentation equilibrium ultracentrifugation the mass of the native enzyme was estimated to be 125 kDa. The N-terminal peptide sequence derived from this protein was used to isolate two very similar cDNA clones encoding novel 500-amino acid proteins, both with calculated molecular masses of 55.9 kDa and p Is of 4.87. The data predict translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation. Deglycosylation assays demonstrate that native PFI from P. filicina is a glycoprotein. The purified protein is chromophoric with a flavin-like UV spectrum and sequence analysis reveals the presence of a flavin-binding motif near the mature N-terminus. Heterologous expression of active PFI in Arabidopsis, using one of the cDNA clones, was successful as evidenced by conversion of arachidonic acid to a conjugated triene in an in vitro assay of the transgenic plant tissues.

A galactose-specific lectin from the red marine alga Ptilota filicina

A lectin from the red marine alga Ptilota filicina (PFL) was isolated by affinity chromatography on cross-linked guar gum. PFL agglutinated native and papain-treated human erythrocytes with preference for type O erythrocytes. The lectin was inhibited by galactose and its derivatives. The most potent inhibitors were p-Nitrophenyl- N-acetyl- α- and β- d-galactosaminide. Porcine stomach mucin, bovine submaxillary gland mucin and asialo bovine mucin were also inhibitory. The M r of PFL, determined by gel filtration, was 56,900. SDS-PAGE gave one band with a subunit M r of 19,320, indicating the native protein to be a trimer of apparently identical subunits. PFL was shown to be rich in acidic and hydroxyl amino acids but low in basic amino acids. The ten N-terminal amino acids were Asx-Thr-Lys-Thr-Tyr-Leu-Leu-Ala-.

Characterization of the substrate binding site of polyenoic fatty acid isomerase, a novel enzyme from the marine alga Ptilota filicina.

The substrate binding site of polyenoic fatty acid isomerase (PFI) has been investigated using a series of alternate substrates and by examination of the pH dependence on the kinetic parameters of PFI with selected substrates. The pH dependence profile of PFI with EPA [(5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid] shows the enzyme to be catalytically active over a wide pH range, with activity being optimal below pH 6.0. Analysis of the kinetic parameters of DHA [(4Z,7Z,10Z,13Z,16Z,19Z)-docosahexen oic acid]; adrenic acid [(7Z,10Z,13Z,16Z)-docosatetraenoic acid]; EPA; arachidonic acid [(5Z,8Z,11Z,14Z)-eicosatetraenoic acid]; anandamide (arachidonyl-N-ethanolamide); and eicosatrienoic acid [(5Z,8Z,11Z)-eicosatrienoic acid] demonstrates that substrates possessing omega-3 olefins (DHA and EPA) have the lowest K(m) values (1.9 and 9.6 microM, respectively). EPA and arachidonic acid showed the highest V(max) values (6.0 and 2.8 micromol min(-1) mg(-1), respectively). The twenty carbon omega-9 fatty acid eicosatrienoic acid showed a relatively large K(m) and had a V(max) approximately 20-fold less than EPA. Anandamide, a substrate analog lacking an ionizable carboxylate, showed a K(m) similar to the other omega-6 fatty acids (arachidonic acid and adrenic acid); however, the V(max) was approximately 5-fold lower than arachidonic acid and 8-fold lower than EPA. Moreover, anandamide demonstrated no pH dependency on its kinetic parameters over a range where EPA showed a 27-fold decrease in V/K(m). NMR spectroscopy was used to determine the structure of the product from reaction of PFI with DHA. These data showed the compound to be (4Z,7Z,9E,11E,16Z,19Z)-docosahexenoi c acid. Reaction of PFI with dihomo-gamma-linolenic acid resulted in the development of two products, one with the characteristic chromophore of a conjugated triene, the other with a chromophore characteristic of a conjugated diene. Analysis of the products from these reactions of PFI, in conjunction with the kinetic parameters from the alternate substrates, provides compelling evidence that the enzyme preferentially orients the substrate in the catalytic site with respect to the methyl terminus.

Biosynthesis of conjugated triene-containing fatty acids by a novel isomerase from the red marine alga Ptilota filicina.

The biosynthesis of conjugated triene-containing fatty acids by the red alga Ptilota filicina is catalyzed by a novel enzyme, polyenoic fatty acid isomerase. The enzyme has been highly purified and is described here for the first time. Matrix-assisted laser-induced desorption mass spectrometry was used to determine that the major protein in the purified enzyme is composed of similar or identical subunits of M(r) 58,119 Da. The native enzyme emerges with an apparent M(r) of 174,000 Da from a gel permeation chromatography column. While this enzyme catalyzes the formation of conjugated trienes from a variety of polyunsaturated fatty acid precursors [arachidonate ((5Z,8Z,11Z,14Z)- eicosatetraenoate) is converted to (5Z,7E,9E,14Z)-eicosatetraenoate; gamma-linolenate ((6Z,9Z,12Z)-octadecatrienoate) is converted to 6Z,8E,-10E-octadecatrienoate], this occurs most rapidly with eicosapentaenoate [(5Z,7E,9E,14Z,17Z)- eicosapentaenoate], which is likely the native substrate. Through a series of experiments utilizing gamma-linolenates stereospecifically labeled with deuterium, we have determined that the enzyme intramolecularly transfers the bis-allylic pro-S hydrogen from the C11 position to the C13 position. Furthermore, the bis-allylic pro-R hydrogen at C8 in gamma-linolenate is lost to the solvent. Using arachidonate as substrate, we demonstrated that the C11 olefinic position becomes protonated by a solvent-derived proton. There appears to be no requirement for molecular oxygen, and the transformation is catalyzed by this single enzyme.

Ptilodene, a novel icosanoid inhibitor of 5-lifoxygenase and Na +/K + ATPase from the red marine alga [formula omitted][formula omitted]J. Agardh

A new fatty acid with novel sites of oxidation was isolated and spectroscopically defined from the red marine alga ptilota filicina (Caramiales). The pure natural product showed inhibitory activity to human 5-lipoxygenase dog kidney Na+/K+ ATPase and the growth of several pathogenic gram positive and negative bacteria.

Two new icosapentaenoic acids from the temperate red seaweed Ptilota filicina J. Agardh.

Two new fatty acid metabolites, 5(Z),7(E),9(E),14(Z),17(Z)-icosapentaenoic acid and 5(E),7(E),9(E),14(Z),17(Z)-icosapentaenoic acid, have been isolated from the temperate red marine alga, Ptilota filicina (Ceramiales, Rhodophyta). The structures of these new compounds, isolated as their methyl ester derivatives, have been deduced from detailed 1H nuclear magnetic resonance (NMR), 13C NMR and 2D-NMR analyses as well as comparisons to known compounds.

Concentration of lodine and bromine by plants in the seas of Japan and Okhotsk

Iodine and bromine content were measured in 24 species of red (Rhodophyta), brown (Phaeophyta) and green (Chlorophyta) seaweeds and 2 species of higher water plants (Embryophyta) from the Sea of Japan, as well as in 12 species of the abovecited taxa and 1 species of flowering plant from the Sea of Okhotsk. Iodine was determined by photometric extraction with brilliant green, and bromine by neutron activation of samples. Phaeophyta and Rhodophyta were richest in iodine and bromine content. Representatives of the order Ceramiales (Rhodophyta) had high iodine and bromine contents. Thus, iodine concentrations in Ptilota filicina, Campylaephora hypnaeoides and Myriogramme yezoensis, a new iodine concentrator discovered by us, amount to 0.42, 0.094 and 0.75%, respectively. Bromine content in representatives of the family Rhodomelaceae was 3.36 and 3.74% in Japan Sea and Okhotsk Sea Rhodomela larix, respectively. Polysiphonia japonica (Rhodomelaceae) is a newly discovered concentrator of bromine (3.20%). Many species of the order Laminariales (Phaeophyta) were characterized by high iodine contents: Laminaria japonica, L. cichoriodes, L. inclinatorhiza, Cymathaere japonica and Alaria marginata. The Br:I ratio for all the species except those that concentrated iodine, was more than 1. Seaweeds that grow at greater depths showed increased iodine and bromine contents. A tendency toward increased iodine content was observed in species growing further to the North. Iodine and bromine were accumulated selectively by various organs of Sargassum pallidum.