Isolation and Some Characterization of an Acidic Polysaccharide with Anti-calcification Activity from Coccoliths of a Marine Alga,Pleurochrysis carterae

Coccolith is a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, and consists of calcium carbonate crystals and organic matrices. EDTA-soluble organic materials extracted from coccoliths of Pleurochrysis carterae showed anti-calcification activity. They were separated by anion-exchange HPLC, and two fractions, fractions A and B, were obtained. Fraction B, which was more active than fraction A, was further separated into six consecutive fractions, B1-B6, by second anion-exchange HPLC. ¹H NMR spectral analyses of these fractions suggested that a novel acidic polysaccharide, designated CMAP, existed throughout B1-B6 and that the latter four fractions mainly contained another acidic polysaccharide, PS-2, characterized previously. Since PS-2 did not show anti-calcification activity, CMAP was found to be the active principle.     

Discrimination of the Cell Surface of the Coccolithophorid Pleurochrysis haptonemofera from Light Scattering and Fluorescence After Fluorescein-Isothiocyanate-Labeled Lectin Staining Measured by Flow Cytometry

We investigated the extent of calcification on the cell surface of the coccolithophorid Pleurochrysis haptonemofera using flow cytometry. Side scattering (SSC) by coccolith-bearing cells was higher than that by naked cells, suggesting the difference was due to scattering of the laser beam by the coccoliths. SSC of coccolith-bearing cells under acidic conditions corresponded well to the extracellular Ca content, although SSC could not be used to detect a delicate change in the coccolith thickness. The increase in SSC during the reproduction of coccoliths after decalcification was consistent with the increase in the number of coccoliths on the cell surface. The fluorescence after fluorescein-isothiocyanate-labeled lectin staining suggests that α-d-mannose, α-d-glucose, d-galactose, d-N-acetylgalactosamine, or derivatives of them are included in the coccoliths. Measurement of SSC and fluorescence after fluorescein-isothiocyanate-labeled lectin staining enabled rapid and quantitative determination of the status on the cell surface and isolation of desirable cells for physiological studies by cell sorting. Received May 22, 2001; accepted July 30, 2001.     

Gene Expression Profiling of Coccolith-Bearing Cells and Naked Cells in Haptophyte <Emphasis Type="Italic">Pleurochrysis haptonemofera</Emphasis> with a cDNA Macroarray System

Pleurochrysis haptonemofera is a unicellular marine coccolithophorid that has calcified scales, coccoliths, on the cell surface. Some coccolithophorids including P. haptonemofera have a coccolith-bearing stage and a naked stage in their life cycles. To characterize genes involved in the coccolithogenesis, we generated a total of 9550 expressed sequence tags (EST) from a normalized cDNA library that was prepared using both coccolith-bearing cells (C-cells) and naked cells (N-cells), constructed a cDNA macroarray using the EST clones, and then analyzed the gene expression specificity in C-cells and N-cells. When cDNA clones whose expression ratio exceeded 3-fold were selected, as many as 180 clones were identified as C-cell-specific ones, while only 12 were found to be N-cell-specific ones. These clones were sequenced, assembled, and homology-searched against a public nonredundant protein database. As a result, they were grouped into 54 C-cell-specific and 6 N-cell-specific genes, and 59% and 50% of these genes exhibited significant similarity to those of other known proteins, respectively. To assess mRNA expression further, Northern hybridization was performed for 12 of the C-cell-specific genes and one of the N-cell-specific ones. These clones, together with the new cDNA macroarray, will provide a powerful tool for the future genome-wide functional analysis of uncharacterized genes related to the regulation of the calcification and life cycle of coccolithophorids. Shoko Fujiwara and Yasutaka Hirokawa contributed equally to this work.     

Effects of Ca and Mg on Growth and Calcification of the Coccolithophorid Pleurochrysis haptonemofera: Ca Requirement for Cell Division in Coccolith-Bearing Cells and for Normal Coccolith Formation with Acidic Polysaccharides

The effects of Ca²⁺ and Mg²⁺ on cellular growth and calcification in Pleurochrysis haptonemofera were investigated. In the presence of a normal concentration of Mg²⁺, coccolith-bearing cells (C-cells) required more than 0.5 mM Ca²⁺ for growth, while naked cells could grow even with 0.5 mM Ca²⁺. The calcification rate of C-cells, which was determined using decalcified cells, was significantly repressed with less than or equal to 0.5 mM Ca²⁺. Although the calcification rate did not change so much with 5–30 mM Ca²⁺, it decreased with higher concentrations of Ca²⁺, as well as C-cell-specific growth repression. Under these conditions, Ca²⁺ affected the rate of coccolith formation, but neither the coccolith morphology nor total amounts and ratios of divalent cations and acidic polysaccharides (Ph-PS-1, -2, and -3) were included in coccoliths. These findings suggest that sufficient calcification is required for the division of C-cells. Under low Ca²⁺ and high Mg²⁺ conditions, coccoliths with an abnormal morphology, having immature shield elements, were synthesized. Composition analysis of the coccoliths revealed high Mg/Ca and low Ph-PS-2/(Ph-PS-1 and -3) ratios, as compared with those under low Ca²⁺ and normal Mg²⁺ conditions, suggesting that the abnormal morphology is due to a change in the crystal type and/or acidic polysaccharide composition.     

Dna Ploidy Studies of Benign and Malignant Tumours: Comparison of Flow Cytometry and Image Analysis Techniques Using Two Types of Cytological Specimen

DNA ploidy studies were carried out on Feulgen stained smears and cytocentrifuge preparations from 35 malignant tumours and four benign neoplasms using the CAS image analyser. The smears were prepared from scrapings from fresh tumour tissue whereas the cytocentrifuge preparations were prepared from single nuclear suspensions from paraffin-embedded cell blocks from the same tumour. Histograms obtained by image analysis of the tumour scrapes were compared with those obtained on the cytocentrifuge preparations. Concordant results were obtained in four benign tumours (100%) and 32 malignant tumours (91%). The results obtained by image analysis were also compared with results obtained by flow cytometry of the tumour tissue. Discordant results were obtained for three malignant tumours. Possible reasons for the discrepancy include sampling error, tumour heterogeneity and selective loss of cell populations during processing.     

D- and L-Aspartic Acids: New Non-sugar Components of Bacterial Polysaccharides

For the first time in bacterial polysaccharides, residues of D- and L-aspartic acids were identified as N-acyl substituents of 4-amino-4,6-dideoxy-D-glucose in the O-antigens of enterobacteria of the genera Providencia and Proteus.     

Protoplast Formation of the Coccolithophorid <Emphasis Type="Italic">Pleurochrysis haptonemofera</Emphasis> in Hypoosmotic K<Superscript>+</Superscript> Solution: Shedding of the Coccosphere and Regrowth of the Protoplast in Normal Medium

Both coccolith-bearing cells (C-cells) and naked cells (N-cells) of the coccolithophorid Pleurochrysis haptonemofera can grow in salinities of more than 7‰ (about 20% of a “normal” sea water salinity [35‰]), with the highest growth rates in salinities of more than 14‰. Microscopic observations of cells suspended in 100 mM NaCl (7‰) showed that, while N-cells were swelling uniformly all over the cell surface, C-cells were bulging the plasma membrane from the hole of the coccosphere at the apical (flagellar) pole of the cell. Effects of several cations and anions on the morphological change of C-cells under hypoosmotic pressure were investigated. When 100 mM K⁺ was used, protoplasts were released from the coccosphere completely in almost all the cells. This phenomenon was shown with K⁺ most effectively. The protoplasts could grow in the fresh medium and form the first coccolith within 9 h.     

Taxonomic Composition and Biological Activity of Microorganisms Associated with a Marine Ascidian Halocynthia aurantium

The taxonomy and biological activity of 176 heterotrophic microorganisms associated with the ascidian Halocynthia aurantiumfrom the coastal waters of Peter the Great Bay (Sea of Japan) were studied. In the microbial community, bacteria of the genera Pseudoalteromonas(38% of all strains studied) and Bacillus(25%) prevailed and showed high biological activity. Bacteria of Pseudoalteromonaswere capable of extracellular hydrolases production and exhibited high antibiotic and antimicrobial properties. Five Pseudoalteromonasstrains were capable of restriction enzyme production. A significant proportion of the pseudoalteromonads inhibited growth ofProteus vulgarisand Candida albicans(81 and 84%, respectively). Spore-forming bacteria of Bacilluswere capable of chitin hydrolysis and extracellular inducible RNAses production (23%). They were active against Staphylococcus aureusand Bacillus subtilis(39 and 23%, respectively). Bacillus pumilusstrain KMM 1364 showed hemolytic activity and antagonistic properties against S. aureusand was found to produce surface active glycolipids and surfactinlike cyclic depsipeptides.     

Comparison of dinucleotide frequency and codon usage inToxoplasma andPlasmodium: Evolutionary implications

Summary The weight-averaged observed/expected dinucleotide frequencies for the sum total of the coding regions of fiveToxoplasma genes were compared with the same parameters previously determined for the coding regions of 21Plasmodium genes. In addition, codon usage in the fiveToxoplasma genes was compared with that in the 21Plasmodium genes, and the percent distribution of amino acids in theToxoplasma protein pool and thePlasmodium protein pool were compared with that in a general protein pool of 314 proteins. The results are consistent with the hypothesis that, contrary to currently held opinion, the generaToxoplasma andPlasmodium are not especially closely related.     

Variation and geographical distribution of ploidy levels inPennisetum sectionBrevivalvula (Poaceae) in Burkina Faso,Benin and southern Niger

Pennisetum sect.Brevivalvula is a species complex characterized by polyploidy and apomixis. Ploidy level was assessed by DAPI-flow cytometry for 304 plants of the section, originating from Burkina Faso, Benin and southern Niger. The results were confirmed for 54 plants based on chromosome counts. The samples show four euploidy levels (with x = 9) distributed among five species:P. hordeoides (2n = 36, 54),P. pedicellatum (2n = 36, 45, 54),P. polystachion (2n = 18, 36, 45, 54),P. setosum (2n = 54), andP. subangustum (2n = 18, 36, 54). The geographical distribution of these ploidy levels seems related to major vegetation zones present in Africa. Diploid populations ofP. polystachion andP. subangustum were found in the Banfora area, in Burkina Faso.     

Acidic protein kinases of Polysphondylium violaceum: Characterization and developmental regulation

Summary The cellular slime mould Polysphondylium violaceum contains two vegetative stage specific acidic (casein) kinases. These two enzymes have been partially purified and their properties investigated. Both utilise casein as their preferred substrate but they can be distinguished in a number of ways, including their responses to spermine, heparin and salt. In addition, they have different affinities for their substrates and different pH activity profiles. It is suggested that they may play a role in a vegetative specific function such as cell division.     

Immunocytochemical Localisation of Glycosaminoglycan-Like Activity in the Mucus and Associated Tissues of Terrestrial Slug Species (Gastropoda: Pulmonata)

Indirect immunofluorescence assays were conducted on cryotome sections of four terrestrial slug species from three distinct phylogenetic groups, Arion ater (L.), Arion hortensis (Férussac), Tandonia (Milax) budapestensis (Hazay), and Deroceras reticulatum (Müller) using monoclonal antibodies for two glycosaminoglycans (GAGs), heparan sulphate, and chondroitin sulphate. Specific staining for a heparan sulphate-like component was demonstrated in the foot and tail regions of each species and was particularly intense in A. ater and A. hortensis, notably in the epidermis and associated mucus-like material, and in mucus-like material from the pedal gland region of the latter species. Subsequent studies with A. ater confirmed the presence of heparan-sulphate–like activity in the caudal gland duct region. No evidence of specific staining for chondroitin sulphate-like activity was found in any of the slug species. This work suggests that a specific GAG, or a group of closely related GAGs, is a common component of mucus in a range of slug species and of different types of mucus, including trail (pedal) mucus and the more viscous mucus produced by the caudal gland.     

Genetic variation and conservation of Changnienia amoena, an endangered orchid endemic to China

Nuclear DNA content (2C) is used as a new criterion to investigate nearly all species of the genus Nerine Herb. The species have the same chromosome number (2n = 2x = 22), with the exception of three triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 18.0–35.3 pg. This implies that the largest genome contains roughly 2 × 10¹⁰ more base pairs than the smallest. The species, arranged according to increasing genome size, fell apart in three groups if growth cycle and leaf width were also considered. A narrow-leafed, evergreen group with a DNA content between 18.0 and 24.6 pg contains thirteen species, a broad-leaved winter growing group with four species has a DNA content from 25.3–26.2 pg and a broad-leafed summer growing group has a DNA content of 26.8–35.3 pg and contains six species. If the presence of filament appendages and hairiness of the pedicels were also considered, the thirteen evergreen species could be further divided into a group without filament appendages or hairy pedicels with a DNA content of 18.0–18.7 pg. A second group without filament appendages but with hairy pedicels had a DNA content of 19.7–22.3 pg. And a third group with both filament appendages and hairy pedicels had a DNA content of 22.0–24.6 pg. The exception is N. marincowitzii that, despite a low DNA content and narrow leaves is summer growing. The broad-leafed group is further characterised by the absence of filament appendages and the absence of strongly hairy pedicels. The exception here is N. pusilla that, despite a high DNA content, has narrow leaves and minutely hairy pedicels. Nuclear DNA content as measured by flow cytometry is shown to be relevant to throw new light on the relationships between Nerine species.     

Transformation and regeneration of loblolly pine: shoot apex inoculation with Agrobacterium

Loblolly pine (Pinus taeda L.) is the mostimportant tree species in US commerce and has much to gain through geneticengineering. This species can be transformed using particle bombardment andAgrobacterium; however, the regeneration of plants fromtransgenic tissues has been difficult and the recovery of transgenic plants hasbeen rare. A shoot-based and genotype-independent transformation methodemploying Agrobacterium tumefaciens was used to facilitaterecovery of plants and permit the transformation of elite germplasm. Shootsfrom4–6 week old seedlings and adventitious shoots from culture wereinoculated with A. tumefaciens EHA101 (pGUS3), or EHA105(pSSLa.3), subjected to selection and regenerated. Shoots that survivedexhibited expression of the uidA gene (GUS) in a patterncharacteristic of the either the CaMV35S promoter (pGUS3), or the larch RbcSpromoter (pSSLa.3) transferred. Recovered plants were screened using PCRamplification. Southern DNA analyses and amplification of the T-DNA borderjunction confirmed genomic integration of both transferreduidA and nptII genes. In this proofofconcept study, the overall recovery of P. taeda shoots wasfair (10–20%), while recovery of intact rooted plants was poor (>1%)due to difficulty in rooting. Recovery of intact rooted plants from inoculatedshoots of P. eldarica and P. radiatawas more efficient (10–30%). The addition of a shoot multiplication stepand effective rooting protocols will improve the efficiency of this genotypeindependent transformation method in P. taeda, and inotherPinus spp.     

Behavioral Responses and Interactions of Estuarine Animals With an Invasive Marsh Plant: A Laboratory Analysis

Invasion by Phragmites australis into tidal marshes previously dominated by Spartina alterniflora is viewed as a serious environmental threat along the Atlantic coast of the US, but little is known about the relative habitat value of the two plants for most estuarine species. This study was designed to investigate behavioral responses, in the laboratory, of three species to the two plants. Fiddler crabs, Uca pugnax, grass shrimp, Palaemonetes pugio, and larval mummichogs (killifish), Fundulusheteroclitus were introduced into aquaria with a bare area, an area with dead Phragmites stems, and an area with dead Spartina stems. All species distributed themselves equally between the Spartina and the Phragmites. The behavior of larval mummichogs in the tanks with predators was observed. In the presence and absence of stems, they utilized the surface of the water as a refuge, as well as the stems, when present. This behavior was equally as effective as being among the stems in promoting larval survival. In microcosms with blocks of marsh with living plants, fiddler crabs and grass shrimp again did not show a preference for either species of plant, while juvenile and adult mummichogs were not consistent. Small fish chose Spartina when in the small microcosm and had no preference in the large one. Large fish chose Spartina in the small microcosm and Phragmites in the large one. Predation by adult mummichogs on grass shrimp was comparable in Spartina and Phragmites microcosms, and predation on tethered shrimp was equivalent in adjacent Spartina and Phragmites marshes in the field.     

Molecular phylogeny of the Acre clade (Crassulaceae): Dealing with the lack of definitions for Echeveria and Sedum

The phylogenetic relationships within many clades of the Crassulaceae are still uncertain, therefore in this study attention was focused on the “Acre clade”, a group comprised of approximately 526 species in eight genera that include many Asian and Mediterranean species of Sedum and the majority of the American genera (Echeveria, Graptopetalum, Lenophyllum, Pachyphytum, Villadia, and Thompsonella). Parsimony and Bayesian analyses were conducted with 133 species based on nuclear (ETS, ITS) and chloroplast DNA regions (rpS16, matK). Our analyses retrieved four major clades within the Acre clade. Two of these were in a grade and corresponded to Asian species of Sedum, the rest corresponded to a European–Macaronesian group and to an American group. The American group included all taxa that were formerly placed in the Echeverioideae and the majority of the American Sedoideae. Our analyses support the monophyly of three genera – Lenophyllum, Thompsonella, and Pachyphytum; however, the relationships among Echeveria, Sedum and the various segregates of Sedum are largely unresolved. Our analyses represents the first broad phylogenetic framework for Acre clade, but further studies are necessary on the groups poorly represented here, such as the European and Asian species of Sedum and the Central and South American species of Echeveria.     

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

Identification and Analysis of Proteins Sharing Dehydroascorbate Reductase Activity

Dehydroascorbate (DHA)-reducing proteins were observed using a native-PAGE/activity staining method, and identified in soybean and rice by Western analysis using antibodies against homologous protein known to catalyse DHA reduction. Administration of 1 mM DHA apparently did not trigger DHA reductase activity in lupin and onion, whereas activity was increased in rice and barley.     

Embryogenesis and genetic stability in long term megagametophyte-derived cultures of larch

Embryogenic cultures derived from megagametophytes of Larix decidua were maintained for up to 17 years. A few lines were divided into sub-lines, which were maintained in the same manner as the others. Embryogenic tissue was grown on 1/2 strength LM medium supplemented with glutamine and casein hydrolysate at constant temperature and light regimes. Chromosome counts were conducted at various times. DNA content was assessed by flow cytometry. Embryogenesis was monitored with each transfer and records of all appearances of green mature embryos were kept. Chromosome number was found to vary. DNA content and chromosome number, both of which had doubled a number of years after initiation, stabilized around 24 chromosomes for most cultures. A few lines showed substantial increases in chromosome number. One of these lines lost vigour and died. Another line showed a further doubling of DNA content. No lines were embryogenic over the entire period. Embryogenicity was lost completely in some lines, but in others the loss was temporary, as periodic restoration of embryogenesis was observed.     

Radiation, multiple dispersal and parallelism in the skinks, Chalcides and Sphenops (Squamata: Scincidae), with comments on Scincus and Scincopus and the age of the Sahara Desert

Phylogenetic analysis using up to 1325 base pairs of mitochondrial DNA from 179 specimens and 30 species of Chalcides, Sphenops, Eumeces, Scincopus and Scincus indicates that Sphenops arose twice independently within Chalcides. It is consequently synonymized with that genus. Chalcides in this broader sense originated in Morocco, diversifying into four main clades about 10 Ma, after which some of its lineages dispersed widely to cover an area 40 times as large. Two separate lineages invaded the Canary Islands and at least five main lineages colonized southern Europe. At least five more spread across northern Africa, one extending into southwest Asia. Elongate bodies with reduced limbs have evolved at least four times in Chalcides, mesic 'grass-swimmers' being produced in one case and extensive adaptation to life in loose desert sand in two others. In clade, Chalcides striatus colonized SW Europe from NW Africa 2.6 Ma and C. chalcides mainland Italy 1.4 Ma, both invasions being across water, while C. c. vittatus reached Sardinia more recently, perhaps anthropogenically, and C. guentheri spread 1200km further east to Israel. C. minutus is a composite, with individuals from the type locality forming a long independent lineage and the remaining ones investigated being most closely related to C. mertensi. In the Northern clade, C. boulengeri and C. sepsoides spread east through sandy habitats north of the Sahara about 5 Ma, the latter reaching Egypt. C. bedriagai invaded Spain around the same time, perhaps during the Messinian period when the Mediterranean was dry, and shows considerable diversification. Although it is currently recognized as one species, the C. ocellatus clade exhibits as much phylogenetic depth as the other main clades of Chalcides, having at least six main lineages. These have independently invaded Malta and Sardinia from Tunisia and also southwest Arabia C. o. humilis appears to have spread over 4000 km through the Sahel, south of the Sahara quite recently, perhaps in the Pleistocene. In the Western clade of Chalcides, C. delislei appears to have dispersed in a similar way. There were also two invasions of the Canary Islands: one around 5 Ma by C. simonyi, and the other about 7 Ma by the ancestor of C. viridanus+C. sexlineatus. C. montanus was believed to be related to C. lanzai of the Northern clade, but in the mtDNA tree it is placed within C. polylepis of the Western clade, although this may possibly be an artifact of introgression. The Eumeces schneideri group, Scincopus and Scincus form a clade separate from Chalcides. Within this clade, the geographically disjunct E. schneideri group is paraphyletic. One of its members, E. algeriensis is the sister taxon to Scincopus, and Scincus may also be related to these taxa. The phylogeny suggests Scincopus entered desert conditions in Africa, up to 9.6 Ma and the same may have been true of Scincus up to 11.7 Ma. Scincus appears to have diversified and spread into Arabia around 6 Ma. Dates of origin and divergence of these skinks, desert Chalcides and other squamates agree with recent geological evidence that the Sahara is at least 5-7 My old. The subspecies Chalcides viridanus coeruleopunctatus is upgraded to the species level as C. coeruleopunctatus stat nov., on the basis of its large genetic divergence from C. v. viridanus.