Bryopsis Plumosa Research Articles

Purification and characterization of a lectin, BPL-3, from the marine green alga Bryopsis plumosa

A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails.

Characterization of carbohydrate combining sites of Bryohealin, an algal lectin from Bryopsis plumosa

Bryohealin is a lectin involved in the wound-healing process of the marine green alga Bryopsis plumosa. In the previous purification study, it has been shown that lectin was composed of two identical subunits of 27 kDa, cross-linked by disulfide bond, and showed binding specificity to N-acetyl-d-glucosamine and N-acetyl-d-galactosamine (GlcNAc and GalNAc, respectively). To determine if the lectin recognize the two different sugars at the same binding domain, the carbohydrate binding sites of Bryohealin was analyzed using chromatography and chemical modification methods. Results showed that the same binding site of the lectin was responsible for the recognition of two sugars, GalNAc as well as GlcNAc. Chemical modification studies showed that hemagglutinating activities of Bryohealin were not affected by modification of histidine, tryptophan, aspartic acid, and glutamic acid. When arginine residues were modified with 1,2-cyclohexanedione, the activity of Bryohealin rapidly decreased. The sugar binding sites remained intact when the lectin was treated with inhibitory sugars (0.2 M GalNAc and/or GlcNAc) prior to 1,2-cyclohexanedione treatment. The sugar binding domain of Bryohealin was predicted from the MALDI-TOF analysis and the full cDNA sequence of the lectin gene.

Purification and characterization of a D‐mannose specific lectin from the green marine alga, Bryopsis plumosa

SUMMARYA d‐mannose specific lectin was purified from the green marine alga, Bryopsis plumosa (Huds.) Ag. The lectin agglutinated horse and sheep erythrocytes. Matrix assisted laser desorption/ionization time of flight mass spectrometry, size exclusion chromatography, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and two dimensional gel electrophoresis (2DE) results showed that the lectin was a monomer with molecular weight of 17 kDa and pI 7.3. The agglutinating activity was inhibited by d‐mannose (1 mM), α‐methyl‐D‐mannose (4 mM) and l‐fucose (8 mM). d‐glucose (125 mM) showed weak inhibition. The lectin did not need divalent cations for agglutinating activity. N‐terminal amino acid sequence of the lectin was analyzed. As the lectin was novel, we named it BPL‐2 (Bryopsis plumosa lectin 2). Full cDNA sequence of BPL‐2 was obtained using cDNA library. It was comprised of 624 bp of open reading frame and 167 bp/57 bp of 3′/5′ untranslated regions as well as N‐terminal signal peptide. No antimicrobial activity of BPL‐2 was observed in four bacteria strains tested.

In vitro antioxidant activity of polysaccharides extracted from Bryopsis plumosa

Polysaccharides extracted from Bryopsis plumosa (Hudson).C. Ag. are a group of sulfated hetero polysaccharides, and their antioxidant activities were investigated employing various established in vitro systems. Available data obtained with in vitro models suggested that among the three samples, B3 (extraction with sodium hydroxide) showed significant inhibitory effects on superoxide radical and DPPH with IC(50) values of 9.2 mu g mL(-1) and 1.7 mg mL(-1); its reducing power was also the strongest among the three samples. These in vitro results clearly establish the possibility that polysaccharides extracted from B. plumosa could be effectively employed as ingredient in health or functional food, to all deviate oxidative stress. However, comprehensive studies need to be conducted in experimental animal models. (C) 2010 Elsevier Ltd. All rights reserved.

Changes in Cytoplasmic Streaming during the Formation of a New Growing Tip in the Coenocytic Green Alga Bryopsis plumosa (Caulerpales, Chlorophyta)

The coenocytic thallus of Bryopsis gametophytes is composed of a main axis and side branches, both of which have a dome-like growing tip with a clear zone of protoplasm and a subsequent cylindrical, basal cell region. Pinnate thallus is formed by regular development of side branches lateral to the main axis. It has been reported that both cytoplasmic streaming and tip growth in the alga are controlled by the microtubule cytoskeleton. We examined the physiology of the tip-growing region in B. plumosa by observing changes in cytoplasmic streaming during 2 different modes of growing tip formation—the germination of spherical cells regenerated from protoplasm aggregates and the formation of side branches. In the main axes, longitudinal cytoplasmic streaming, indicating chloroplast movement, was evaluated using time-lapse videomicroscopy. In the spherical cells regenerated from protoplast aggregates, chloroplasts showed active movement before and after their germination, but showed just small wriggling motion during germination. Chloroplast motility was unchanged during the early stages of side branch formation, during which a clear zone developed in cell surface protrusion. Thus cytoplasmic streaming appeared not to be involved in the growing tip formation in B. plumosa and hence microtubules may play a role in the growing-tip morphogenesis in this alga via mechanisms not directly related to the cytoplasmic streaming.

Epiphytism of Gracilaria cliftonii (Withell, Millar & Kraft) from Western Australia

Epiphytism in Gracilaria is a common phenomena observed in natural populations and under culture conditions. Generally, epiphytes are attached superficially to the surface of the host however, genera such as Polysiphonia spp. and Ceramium spp. can penetrate into the host tissue affecting its growth and hence productivity. The present paper aims to identify and quantify epiphyte abundance and characterise their attachment and penetration on the natural populations of Gracilaria cliftonii collected from Shoalwater Marine Park, Perth, Western Australia. G. cliftonii samples were collected monthly for 1 year and their epiphytes were identified. Histological studies of G. cliftonii were also performed on epiphyte attachment sites. Twenty-four species of macroalgae epiphytes were recorded, 21 belonging to Rhodophyta and 3 to Chlorophyta. Hypnea episcopalis and Polysiphonia forfex were the dominant (p < 0.05) species during the sampling period. The maximum epiphytic load was observed in October (40%), however, the maximum diversity of epiphytes was recorded in September and February (n = 14). Light and scanning electron microscopy studies of the epiphyte attachment sites revealed that P. forfex and Ceramium isogonum penetrated into the cortex and outer medulla of G. cliftonii, while Bryopsis plumosa and Laurencia clavata altered the cellular structure of the cell wall of G. cliftonii.

Influence of macroalgal diversity on accumulation of radionuclides and heavy metals in Bulgarian Black Sea ecosystems

Radionuclides and heavy metals were studied in green, brown and red Black Sea macroalgae by low-level gamma spectrometry and atomic absorption spectrometry. The samples were collected along the whole Bulgarian coast from 1996 to 2004. The levels have been depending on algae species, locations and year of sampling. The highest 137Cs levels were found in red Ceramium rubrum species from all studied locations, while 226Ra and 210Pb were up to three orders of magnitude higher in Bryopsis plumosa. The data showed that the red algae species (Rhodophyta) accumulate more heavy metals than the other phyla (except for Fe whose values were higher in green algae). The data confirmed that algae are valuable indicators of the environmental contamination. The observed elevated levels were mainly due to Danube, Dnieper and Dnester inputs in the NW corner of the Black Sea.

Antimicrobial activity of seaweeds extracts against multiresistant pathogens

Fourteen seaweeds collected from the intertidal zone of Southwest coast of India were tested against ten human pathogen bacteria and one human pathogen fungus using the well diffusion test in the casitone agar medium. The species used in the present study include five Chlorophyta (Bryopsis plumosa, Ulva fasciata, Acrosiphonia orientalis, Chaetomorpha antennina, Grateloupia filicina), five Rhodophyta (Hypnea pannosa, Gracilaria corticata, Centroceras clavulatum, Portieria hornemannii, Cheilosporum spectabile) and four Phaeophyta (Padina tetrastromatica, Sargassum wightii, Stocheospermum marginatum, Chnoospora bicanaliculata). Of these, seven species were determined to be highly bioactive and screened on the multiresistant pathogens. We found that drying process has eliminated the active principles in the seaweeds. In the present study, methanol:toluene (3∶1) was found to be the best solvent for extracting the antimicrobial principles from fresh algae. However, the ethanolic extract showed no antibacterial activity.Acrosiphonia orientalis showed activity against 70% of the tested organisms.Stocheospermum marginatum was the only seaweed that showed activity againstKlebsiella pneumoniae. The extract fromGracilaria corticata was highly active againstProteus mirabilis, a Gram negative pathogenic bacterium. The present findings revealed that the tested seaweeds were highly active against Gram negative bacteria than Gram positive bacteria. The antimicrobial principle from seaweed was found to be a lipophilic compound. The compound was stable over a wide range of temperature (30–60 °C). The active principles of highly active seaweedsAcrosiphonia orientalis andStocheospermum marginatum were bactericidal.

Equal Sex Ratios of a Marine Green Alga, Bryopsis plumosa

By finding some important culture conditions as below, we succeeded in experimentally controlling the whole life history of a dioecious marine green alga, Bryopsis plumosa (Hudson) C. Agardh. In this study, we focused on the primary and secondary sex ratios (i.e. at inception and maturity) using these culture techniques. Gametogenesis was induced by culturing haploid gametophytes with Provasoli's enriched seawater (PES) medium under a 14:10 h light:dark cycle at 14 degrees C. Formed zygotes grew into diploid sporophytes, which were cultured for 3 months with PES medium under a 14:10 h light:nbsp;dark cycle at 18 degrees C. Then they were transferred into Schreiber medium and cultured under a 10:14 h light:dark cycle at 22 degrees C. Within 1 week, zoosporogenesis was observed. Zoospores were released within a couple of days. Each zoospore soon germinated and grew into a unisexual gametophyte. The primary sex ratio was examined in gametophytes that originated from a single sporophyte. The secondary sex ratio was studied in the field. Both were estimated as 1:1. Synchronized meiotic cell divisions might occur during zoosporogenesis dividing each sex-determining factor evenly among zoospores. Given the equal sex ratio at maturity, there seems to be no environmental factor that differentially affects the survival of male or female gametophytes in nature.

MOLECULAR CHARACTERIZATION OF THE LECTIN, BRYOHEALIN, INVOLVED IN PROTOPLAST REGENERATION OF THE MARINE ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA)(1).

When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5' untranslated region (UTR) and 201 bp of 3' UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C-terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.

Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia

Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.

Newly metamorphosed Elysia clarki juveniles feed on and sequester chloroplasts from algal species different from those utilized by adult slugs

The adult, sacoglossan sea slug, Elysia clarki (Pierce et al. in Molluscan Res 26, 2006), sequesters functional chloroplasts in cells of the digestive diverticula from four species of macroalgae in the order Bryopsidales (Penicillus capitatus, Penicillus lamourouxii, Halimeda incrassata, and Halimeda monile). Feeding experiments were conducted in December, 2003, using individuals raised in the laboratory from egg masses laid by E. clarki adults which had been collected from Grassy Key, Florida, USA, and 29 species of macroalgae collected from the Florida Keys, Tampa Bay, Tarpon Springs, Florida, or Woods Hole, Massachusetts or obtained from the Culture Collection of Algae at the University of Texas at Austin, Texas. For the first 14-day post-metamorphosis, juveniles ate only the thin filamentous species, Bryopsis plumosa or Derbesia tenuissima. Transmission electron microscopy showed that the chloroplasts from both algae were sequestered intracellularly in juvenile slugs. Individuals offered any other macroalga, including the four species fed on by adults, did not feed on or incorporate any chloroplasts, and soon died. Juveniles switched from B. plumosa to P. capitatus at a length of ∼ 1.0 cm, and fixed for microscopy 14 days later had intact intracellular chloroplasts from both algae. Nucleotide sequences of the chloroplast gene, rbcL, from DNA extracted from E. clarki, collected from Vaca Key, Florida, were aligned with 22 other available macroalgal rbcL sequences indicating that these E. clarki adults had fed on three species of algae in the Bryopsidales including Bryopsis pennata, and TEM results partially confirmed this conclusion.

Macroalgal distribution patterns in a shallow, soft-bottom lagoon, with emphasis on the nonnativeGracilaria vermiculophylla andCodium fragile

We determined the distribution of macroalgae in Hog Island Bay, a shallow coastal lagoon in Virginia, USA, seasonally at 12 sites from 1998 to 2000 and at 3 representative sites from 2000 to 2002. We analyzed macroalgal biomass, taxonomic richness, and abundance of two non-native species, the cryptic invaderGracilaria vermiculophylla and the conspicuousCodium fragile, with respect to season, location (mainland, mid lagoon, barrier island sites), and elevation (intertidal, subtidal). Taxonomic richness, total algal biomass, and nonnative biomass peaked in the summer months when temperature and light availability were highest. A few stress tolerant and ephemeral algae dominated the algal assemblage.G. vermiculophylla constituted 74% of the entire algal biomass, was the most abundant alga in all seasons, locations, and elevation levels, and was positively correlated with taxonomic richness and abundance of filamentous species.Ulva curvata, Bryopsis plumosa, andC. fragile accounted for an additional 16% of the algal biomass. There are distinct habitats in Hog Island Bay that can be classified into low diversity-low biomass regions near the mainland and barrier islands and high diversity-high biomass regions in the open mid lagoon, where abundant shells for attachment and intermediate levels of water column nutrients and turbidity likely create better growth conditions. Taxonomic richness and biomass were higher in subtidal than intertidal zones, presumably due to lower desiccation stress. This study provides an example of how a single invasive species can dominate an entire assemblage, both in terms of biomass (being most abundant in all seasons, locations, and tidal levels) and species richness (correlating positively with epiphytic filamentous taxa). By adding hard-substratum structural complexity to a relatively homogenous soft-substratum system,G. vermiculophylla increases substratum availability for attachment and entanglement of other algal species and enhances local diversity. Without widespread and abundantG. vermiculophylla, taxa likePolysiphonia, Ceramium, Bryopsis, Ectocarpus, andChampia would likely be much less common. This study also highlights the importance of using DNA analysis of voucher specimens in monitoring programs to accurately identify cryptic invaders.

PURIFICATION AND CHARACTERIZATION OF A LECTIN, BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) 1

When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin–carbohydrate complementary system. Two sugars, N‐acetyl‐d‐glucosamine and N‐acetyl‐d‐galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate‐labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N‐acetyl‐d‐galactosamine and N‐acetyl‐d‐glucosamine in the crude extract. One‐step column purification using N‐acetyl‐d‐glucosamine‐agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross‐linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The N‐terminal 15 amino acid sequence of the lectin was Ser–Asp–Leu–Pro–Thr–X–Asp–Phe–Phe–His–Ile–Pro–Glu–Arg–Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa“bryohealin.”

Susceptibility of macroalgae to herbivorous fishes at Rocas Atoll, Brazil

Although herbivory by fishes is recognized as a funda- mental process structuring coral reef communities, the susceptibility of macroalgae and potential effects of this process at Rocas Atoll are unknown. Macroalgal con- sumption was evaluated through field transplant assays onto reef sites to investigate the differential susceptibility of species to herbivory by fishes. Of 13 species exam- ined, the red macroalga Digenea simplex was highly pre- ferred and probably constitutes the most important food resource to herbivorous fishes in almost all parts of the atoll. The green macroalga Bryopsis plumosa and the brown algae Dictyopteris jamaicensis, Dictyota crispata, D. ciliolata, D. cervicornis, D. menstrualis, D. mertensii, D. pfaffii and D. pinnatifida were less preferred, probably due to chemical defenses, since all are known to produce secondary metabolites, many of which are broad spec- trum feeding deterrents against herbivores including reef fishes. Finally, the algae Sargassum polyceratium and Gelidiella acerosa were less preferred, probably because of both morphological features and chemical defenses. This differential susceptibility of the seaweeds to herbi- vory associated with different spatial herbivory pressure may be responsible for the variable distribution and abundance of seaweeds around the Rocas Atoll reef.

The Intracellular, Functional Chloroplasts in Adult Sea Slugs (Elysia crispata) Come from Several Algal Species, and are Also Different from those in Juvenile Slugs.

The sacoglossan sea slug, Elysia crispata, sequesters chloroplasts from its algal food source within specialized cells lining the digestive diverticulum. These stolen chloroplasts photosynthesize within the slug cell cytoplasm as long as four months--one of the longest kleptoplastic associations known [1]. While many other sacoglossan species feed on and sequester chloroplasts from only one species of algae, adult E. crispata sequester plastids from three different species of algae; Penicillus capitatus, Halimeda incrassata, and Halimeda monile [2]. We have now done feeding experiments testing the ability of newlymetamorphosed, juvenile E. crispata, raised from egg masses in the lab, to sequester chloroplasts from multiple algal species using a large range of potential algal food sources. Surprisingly, juvenile E. crispata fed on different algal species (Bryopsis plumosa and Derbesia tenuissima) from those utilized for sources of symbiotic plastids in the adults. Transmission electron microscopy (TEM) verified that the B. plumosa and D. tenuissima chloroplasts were sequestered intracellularly in the juvenile slugs. In addition, juvenile E. crispata fed exclusively on B. plumosa could be grown to adult size, and, as adults, they would switch to feeding on Penicillus capitatus if presented with it. Since the fine structure of B. plumosa and P. capitatus chloroplasts are easily distinguishable, TEM indicated that both types of chloroplasts are sequestered simultaneously inside the same cell in animals fed on both species of algae (Fig. 1). Finally, a newly discovered population of E. crispata which lives in an area where only B. plumosa is present showed the presence of B. plumosa chloroplasts sequestered in adult slug digestive cells using TEM analysis and using molecular markers. Adult slugs fed on B. plumosa in the lab maintained chloroplasts for approximately as long as the field-collected animals. These results indicate that E. crispata not only eats several species of algae, but also is capable of maintaining symbiotic plastids concurrently from those species for long periods.

Behavior of nuclei during zoosporogenesis in Bryopsis plumosa (Bryopsidales, Chlorophyta)

The behavior of nuclei during zoosporogenesis in Bryopsis plumosa (Bryopsidales, Chlorophyta) was examined by fluorescence and electron microscopy. Each mature filamentous sporophyte had a single lenticular nucleus, which was about 25 microm in diameter and embedded in a thick cytoplasmic layer. At the commencement of multinucleation, giant nuclei with large vacuolated nucleoli, giant nuclei containing chromosomes, and dumbbell-shaped nuclei were observed. Sometimes, two small nuclei also appeared in the thick cytoplasm where the giant nucleus had presumably been present. Electron microscopy revealed the existence of ribbon-like structures resembling synaptonemal complexes within the nucleus having a large vacuolated nucleolus. Nuclei extended their distribution by repetitive divisions. A pair of centrioles was adjacent to the interphase nucleus. When the nuclei were distributed throughout the cell, they became localized nearly equidistantly from one another, each being surrounded by several chloroplasts. At this stage, many centrioles lay along the nuclear surface. The bulk of cytoplasm was then divided into many masses of protoplasm, each of which developed into a uninucleate, stephanokontic zoospore with a whorl of flagella.

Metabolic and enzymatic activities of individual cells, spheroids and embryos as a function of the sample size

Respiration, photosynthesis and peroxidase activities of living spherical samples, such as algal protoplasts, breast cancer spheroids and bovine embryos, were characterized with scanning electrochemical microscopy (SECM). The concentration profile of the metabolic product around the spherical sample was directly measured by scanning with a probe microelectrode. According to the spherical diffusion theory, the total mass transfer rate per spherical sample is linear to the multiplication of the sample radius and the concentration difference between the sample surface and the bulk of the solution. Therefore, the sample radius is a key parameter to assess the viability of the living samples. For example, we investigated the respiration and photosynthesis activities as a function of the size of the protoplast ( Bryopsis plumosa). The respiration rate was linear to the cube of the sample radius. On the contrary, the photosynthesis rate was linear to the square of the sample radius, suggesting that the former is controlled by the volume of the protoplast, and the latter is controlled by the surface area of the protoplast. We will also discuss the size-dependent activity of the breast cancer spheroids and the bovine embryos. Furthermore, relations between the sample size, the concentration difference, and the oxygen consumption rate of the cryo-preserved bovine blastocysts were investigated.

Novel sporophyte-like plants are regenerated from protoplasts fused between sporophytic and gametophytic protoplasts of Bryopsis plumosa

Protoplasts of the marine coenocytic macrophyte Bryopsis plumosa (Hudson) C. Agardh. [Caulerpales] can easily be obtained by cutting gametophytes or sporophytes with sharp scissors. When a protoplast isolated from a gametophyte was fused with a protoplast isolated from a sporophyte of this alga, it germinated and developed into either one of two completely different forms. One plant form, named Type G, appeared quite similar to a gametophyte, and the other, named Type S, looked similar to a sporophyte. While the Type G plant contained many small nuclei of gametophyte origin together with a single giant nucleus of sporophyte origin, the Type S plant contained many large nuclei of uniform size. These large nuclei in the Type S plant had metamorphosed from the gametophytic nuclei, and were not formed through division of the giant nucleus of sporophyte origin. Fragments of the Type S plant, each having such a large nucleus, developed into creeping filaments that look very similar to sporophytes. While cell walls of gametophytes and Type G plants were stained by Congo-red, those of the thalli of regenerated Type S plants and sporophytes were not stained by the dye. This indicated that the large nuclei of the Type S plant did not express genes for xylan synthesis, which are characteristic of gametophytes. Two-dimensional gel electrophoretic analysis revealed that most of the proteins synthesized in the Type S plant were identical to those of sporophytes. These results strongly suggest that in the Type S plant, the gametophytic nuclei are transformed into sporophyte-like nuclei by an unknown factor(s) produced by the giant nucleus of sporophyte origin and that the transformed nuclei express the set of genes characteristic of sporophytes. Despite morphological similarity, however, the regenerated Type S plant could not produce zoospores, because its large nuclei did not divide normally. The transformed large nuclei of gametophyte origin still seemed to be in the haploid state.

Placidenes C-F, novel alpha-pyrone propionates from the Mediterranean sacoglossan Placida dendritica.

Four new alpha-pyrone-containing propionates (5-8) and an unprecedented hydroperoxide 9 have been isolated from the mantle extract of Placida dendritica, a Mediterranean sacoglossan that lives upon the green alga Bryopsis plumosa. The new metabolites co-occur with the related compounds 1-4, which have been described in previous studies of the mollusc. The presence of 9 opens intriguing perspectives on the ecological role of placidenes. This paper reports the isolation and structural elucidation of the new compounds 5-9.