Polysaccharides from Ulva pertusa (Chlorophyta) and preliminary studies on their antihyperlipidemia activity

Polysaccharides from Ulva pertusa were isolated and prepared by extraction in hot water and precipitation by ethanol. The water-soluble polysaccharides were chemically well defined, containing 47.0% total carbohydrate, 23.2% uronic acids, 17.1% sulfate groups, 1.0% N and 29.9% ash. Gas chromatography analysis demonstrated that the neutral sugars were mainly composed of rhamnose, xylose and glucose and smaller amounts of mannose, galactose and arabinose. The FTIR and ¹³C-NMR spectra indicated that basic repeating units of the polysaccharides were (-D-GlcpA-(1-> 4)--L-Rhap 3S) and (-L-IdopA-(1-> 4)--L-Rhap 3S). Fifty ICR mice were used to study the effect of water-soluble polysaccharides from Ulva pertusa on the level of plasma lipids, with inositol niacinate as positive control. The results indicated that the polysaccharides significantly lowered the contents of plasma total cholesterol, low-density lipoprotein cholesterol, triglyceride and markedly increased the contents of serum high-density lipoprotein cholesterol, compared with the hyperlipidemia control group (p > 0.01). Moreover, administration of polysaccharides significantly decreased the atherogenic index. The present results suggest that the polysaccharides from Ulva pertusa have great potential for preventing ischemic cardiovascular and cerebrovascular diseases.     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta, Ulvales)

A simple enzyme mixture containing 2% Cellulase Onozuka R–10 and1% Macerozyme R–10 prepared in deionised water supplemented with 3% NaCland 1 mM CaCl₂ was developed for isolating rapidlyprotoplasts from different species of Monostroma,Enteromorpha and Ulva. The yield fordifferent species of Monostroma ranged from 9.6 ×10⁶ to 10.2 × 10⁶ cells g^–1f. wt thallus, and forEnteromorpha from 3.48 × 10⁶ to 11.7× 10⁶ cells g^–1 f. wt and forUlva from 4.58 × 10⁶ to 26.8 ×10⁶ cells g^–1 f. wt. The overallregeneration rate of the protoplasts isolated was usually > 90% and showednormal morphogenesis. The method yields rapid mass production of viableprotoplasts with high regeneration rates.     

Isolation of Pollutant (Pine Needle Ash)-Responding Genes from Tissues of the Seaweed Ulva Pertusa

Genetic responses of the seaweed Ulva pertusa to pine needle ash have been compared using differential display technique. The tissue viability was assessed to evaluate the stress level with triphenyltetrazolium chloride. Total RNA, from tissues treated in seawater containing ash, was reverse transcribed and amplified by PCR with arbitrary primers. The genetic fragments responding to the stress were selectively isolated from agarose gel and sequenced with a DNA auto sequencer. According to sequence analysis, an ash-inducible gene (342 bp) and an ash-suppressed gene (1690 bp) were identified as hypothetical proteins.     

Ulva rigida (Ulvales,Chlorophyta) tank culture as biofilters for dissolved inorganic nitrogen from fishpond effluents

Ulva rigida was cultivated in 7501 tanks at different densities with direct and continuous inflow (at 2, 4, 8 and 12 volumes d^–1) of the effluents from a commercial marine fishpond (40 metric tonnes, Tm, of Sparus aurata, water exchange rate of 16 m³ Tm^–1) in order to assess the maximum and optimum dissolved inorganic nitrogen (DIN) uptake rate and the annual stability of the Ulva tank biofiltering system. Maximum yields (40 g DW m^–2 d^–1) were obtained at a density of 2.5 g FW 1^–1 and at a DIN inflow rate of 1.7 g DIN m^–2 d^–1. Maximum DIN uptake rates were obtained during summer (2.2 g DIN M^–2 d^–1), and minimum in winter (1.1 g DIN m^–2 d^–1) with a yearly average DIN uptake rate of 1.77 g DIN m^–2 d^–1 At yearly average DIN removal efficiency (2.0 g DIN m^–2 d^–1, if winter period is excluded), 153 m² of Ulva tank surface would be needed to recover 100% of the DIN produced by 1 Tm of fish.Abbreviations DIN= dissolved inorganic nitrogen (NH _inf4 ^sup+ + NO _inf3 ^sup– + NO _inf2 ^sup– ); - FW= fresh weight; - DW= dry weight; - PFD= photon flux density; - V= DIN uptake rate     

Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales,Chlorophyta)

Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15–20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 × 10⁵ cells ml⁻¹ before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10–15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 μs duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18–24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.     

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae)

Protoplast were isolated from a filamentous brown alga, Sphacelaria sp. (Sphacelariales, Phaeophyta), using alginate-lyases extracted from marine molluscs, and commercial pectinase and cellulase. Yields were about 4000 protoplasts per gram of fresh tissue. Different types of protoplasts, originating from apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells produced a higher percentage of protoplasts (approx. 2%), compared with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types of protoplasts divided at least once in culture, but did not develop further.     

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 10⁶protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.     

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, <Emphasis Type="Italic">Ulva pertusa</Emphasis> (Chlorophyta)

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl⁻, as confirmed by the use of the Cl⁻ channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl⁻ permeable channels are responsible for the influx of Cl⁻ into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.     

Chemical characterisation and gelling properties of cell wall polysaccharides from species of Ulva (Ulvales,Chlorophyta)

Two alkali-soluble polysaccharide fractions from the cell wall of Ulva rigida were determined to be ,-1,4-linked glucoxylans and a -1,4-linked glucuronan by chemical and NMR spectroscopic analysis. The ¹³C NMR spectrum of water-soluble xyloglucuronorhamnan sulfate from Ulva rigida referred to as ulvan is reported and the ¹³C and ¹H NMR chemical shifts of its major repeating unit, the aldobiuronic acid -D-GlcA-(1,4)-L-Rha, are given. The composition and gelling properties of ulvan from Ulva species from green tides are also reported. The thermoreversible gel required both calcium and borate ions and the shear storage modulus G was ion concentration dependent. The mechanism of gelation and the associations of the different Ulva cell wall soluble polysaccharides are discussed.     

Plant regeneration from protoplasts isolated from cotyledons of Sesbania bispinosa

Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d, 2 mg l^-1 benzyladenine (BA), 1 mg l^-1 glutamine and 0.5 and formed callus. The first division occurred after 3–4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l^-1 indolebutyric acid (IBA) and 1 mg l^-1 BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l^-1 IBA.     

cDNA cloning of a lectin-like gene preferentially expressed in freshwater from the macroalga Ulva limnetica (Ulvales, Chlorophyta)

The macroalgal species Ulva limnetica Ichihara et Shimada was investigated to understand the molecular mechanism of its tolerance or adaptation to freshwater. We detected a 19 kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which accumulated in greater amounts in freshwater conditions compared with marine conditions. The band was excised and the partial amino acid sequence was determined by Edoman degradation. Based on the sequences, we isolated the corresponding cDNA by the rapid amplification of cDNA ends (RACE) technique. The constructed, full-length cDNA was 1272 bp in length, consisting of 198 bp 5'-non-coding region, an open reading frame of 840 bp (279 amino acids), 233 bp 3'-non-coding region and poly (A) tail. The protein encoded by the cDNA showed 30% identity and 45% similarity to lectin isolated from Ulva pertusa Kjellman, and we named this gene ULL (encoding Ulva limnetica lectin-like protein). Northern blot analysis demonstrated that the expression level of the ULL in the freshwater-cultured sample was higher than in the seawater-cultured sample.     

Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta)

The chemical composition and structures of several ulvan extracts isolated from various Ulva species were studied. They were all composed mainly of rhamnose, glucuronic acid, xylose, glucose and sulphate with smaller amounts of iduronic acid and traces of galactose. Proteins were also present, most likely as contaminants. Precise quantification of the uronic acid content by chemical-enzymatic hydrolysis coupled to HPAEC-PAD analysis and by colorimetry was not achieved, most likely due to the incomplete hydrolysis of glucuronan segments, inadequate HPAEC-pulsed-amperometric response factor for iduronic acid and to a possible differential colorimetric response of the two uronic acids. ¹³C NMR spectroscopic investigation of different ulvans demonstrated that they were all based on ulvanobiuronic acid 3-sulphate A and B repeating units [β-D-Glc pA-(1->4)-α-L-Rhap3S and α-L-IdopA-(1->4)-α-L-Rha p3S, respectively] as well as contiguous β 1->4 linked D-glucuronic acids possibly occurring either in ulvan or as a separate glucuronan. Marked variations in the content of the repeating structures were seen among the different samples. However, due to the limited number of samples studied, no conclusion was reached concerning the effects of species and ecophysiological conditions on the chemistry of ulvan. This revised version was published online in June 2006 with corrections to the Cover Date.     

The induction of reproductive cell formation of Ulva pertusa Kjellman (Ulvales, Ulvophyceae)

Synchronous zooid formation in Ulva pertusa Kjellman was induced in excised disks maintained in sterilized seawater at 20°C, 12:12 h L:D cycle and fluorescent light at 100 μmol photons m ²s ¹. Zooids were reieased from mature disk tissue on the morning of the second or the third day after excision. The degree of zooid formation was found to be dependent on disk size and the region of the mother thalius from which the disk tissue was excised. Zooid formation was induced in more than 90% of small disks (0.9 mm in diameter) which were taken from the margins of the Ulva thalli. When disks were incubated together with a perforated mother thalius, the disks remained sterile. The presence of maturation inhibitors in vegetative thalli is suggested.     

Algicidal activity of polyunsaturated fatty acids derived from <Emphasis Type="Italic">Ulva fasciata</Emphasis> and <Emphasis Type="Italic">U. pertusa</Emphasis> (Ulvaceae,Chlorophyta) on phytoplankton

Isolation of algicidal compounds from Ulva fasciata revealed that the algicidal substances were the polyunsaturated fatty acids (PUFAs) as hexadeca-4,7,10,13-tetraenoic acid (HDTA) C16:4 n-3, octadeca-6,9,12,15-tetraenoic acid (ODTA) C18:4 n-3, α-linolenic acid (ALA) C18:3 n-3 and linoleic acid (LA) C18:2 n-6. The fatty acid composition of four species of Ulvaceae (U. fasciata, U. pertusa, U. arasakii and U. conglobota) was analyzed by capillary gas chromatography to investigate the relationship with the algicidal activity. The results indicate that highly algicidal species, U. fasciata and U. pertusa, showed higher contents of C16:4 n-3, C18:3 n-3, and C18:4 n-3. Concentrations of these PUFAs released from the seaweed in the culture medium were also analyzed. These PUFAs were found to be significantly active against Chattonella antiqua, C. marina, Fibrocapsa japonica, Heterosigma akashiwo, Karenia mikimotoi, moderately effective against Heterocapsa circularisquama, Prorocentrum minimum, P. sigmoides, Scrippsiella trochoidea, whereas low effective against Alexandrium catenella and Cochlodinium polykrikoides. It is suggested that the PUFAs are useful mitigation agents to remove several harmful effects without causing detrimental effects on surrounding marine living organisms.     

Plant regeneration fromBrassicanigra (L.) Koch stem protoplasts

Summary Protoplasts ofBrassica nigra (L.) Koch were isolated from stem peels of bolting racemes and cultured in 1.5 ml of VN1 liquid medium. The protoplasts in the liquid medium were plated on top of half strength MS medium supplemented with 400 mg/liter glutamine, 15 mg/liter glutathione, 50 mg/literl-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 mg/liter 2,4-dichlorophenoxyacetic acid, 1.5% sucrose, and 5% mannitol, pH, 5.7, solidified with 0.3% agarose. Ten percent of calli obtained from the protoplasts developed into plantlets within 4 wk after transfer onto 2N regeneration medium which contains MS salts plus 200 mg/liter casein hydrolysate, 0.625 mg/liter 6-benzylaminopurine, 0.625 mg/liter kinetin, 0.625 mg/liter 6-(γ,γ-dimethylallylamino)-purine, 0.625 mg/liter zeatin, 0.5 mg/liter 1-naphthaleneacetic acid, 1.5% sucrose, and 0.4% agarose. THis is the first report of plant regeneration fromB. nigra protoplasts.     

Optimization of formation and regeneration of protoplasts from biocontrol agents ofTrichoderma species

The optimal conditions necessary for a large yield and a high frequency of regeneration of protoplasts isolated from the biocontrol agentsTrichoderma koningii andT. harzianum were investigated. Protoplast yields were 1.2×10⁸/ml fromT. koningii and 6×10⁷/ml fromT. harzianum when 20-h mycelial culture was treated with a lytic enzyme solution containing Novozym 234 (15 mg/ml), sucrose (0.6 M) and citrate phosphate buffer (0.02 M), pH 5.6 at 31°C. When the protoplasts were grown in the regeneration medium containing yeast extract (1.5%), 1 I of Mandel's salts, pH 5.6, and glucose (0.8 M), a high frequency of regeneration of the protoplast was obseved: 66% forT. koningii and 45% forT. harzianum. Two patterns of regeneration were observed. First, the hyphae arose directly from the regenerated protoplast mother cell. Second, a chain of bud cells developed from the protoplast and subsequently generating hyphae generally protruded from the terminal bud cells.     

New species of freshwater Ulva, Ulva limnetica (Ulvales, Ulvophyceae) from the Ryukyu Islands, Japan

Ulva limnetica Ichihara et Shimada, sp. nov. (Ulvales, Ulvophyceae) is described from the Ryukyu Islands, Japan, and is characterized by thalli that are: (i) branched, tubular, fragile and wrinkled; (ii) up to 80 cm in height and up to 2 cm in diameter; (iii) light to yellowish green in color; and (iv) having an asexual reproduction by means of quadriflagellate swarmers. Rhizoidal cells bear tubular extensions on the outside of the cell layer in the stipe. Ulva limnetica is distinguished from species with similar thalli by chloroplast disposition, branching pattern, number of pyrenoids per cell and gross morphology. It is also distinguished by sequences of the nuclear-encoded 18S ribosomal RNA gene, internal transcribed spacer 2 region and the plastid-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxgenase gene ( rbc L). Ulva limnetica was clustered with other Ulva species in an early diverging lineage within the genus.     

Genetic diversity and biogeography of native and introduced populations of Ulva pertusa (Ulvales,Chlorophyta)

Genetic diversity of native and introduced populations of Ulva pertusa (Ulvales, Chlorophyta) was examined using genetic markers of chloroplast, mitochondria and nuclear non‐coding region sequences. In the preliminary investigations to genetically identify the species for further analyses, U. pertusa was found only from temperate coasts of the more extensive collection sites including tropical coasts suggesting that it is a temperate species and basically not distributed in tropical regions. For chloroplast and mitochondrial sequences, repeating patterns of short tandem repeat sequences and nucleotide substitutions were used to recognize the haplotypes (genetic types). A total of 48 haplotypes based on combinations of chloroplast and mitochondrial haplotypes were recognized in the 244 specimens collected in the presumptive native range (Northeast Asia) and introduced populations (North America, Australia, New Zealand, Chile and Europe). Among them, 46 haplotypes (H1–H8 and H11–H48) were recognized in Northeast Asia, whereas only 1–5 haplotypes were recognized in the other areas. Nuclear microsatellite sequences were also analyzed. The lengths of the PCR products including the nuclear microsatellite region of 234 specimens were determined, and a total of 17 genotypes were recognized. Among them, 14 genotypes were found in Northeast Asia, whereas 1–7 genotypes were recognized in the other areas. Based on the results, the hypothesis that the native range of the species is in Northeast Asia was supported, and the populations outside this range were concluded to be non‐indigenous populations.