THE ULTRASTRUCTURE OF SEPARATED AND CULTURED CELL OF PORPHYRA YEZOENSIS

There are many reports that cells (protoplasts) separated from the thallus of Porphyra by enzyme can develop to normal leafy thalli in the same way as monopores. But there are few investigations on the subcellular structure of the isolated vegetative cell for ccmparison with the subcellular structure of monospores. To clarify whether the separated and cultured cells undergo the same or similar ultrastructure changes during culture and germination as monospores undergo in their formation and germination, we observed their ultrastructure, ccxnpared them with those of the monospore and found that the ultrastructure of separated and cultured cells did not have the characteristic feature as that of monospore formarion, such as production of small and large fibrous vesicles, but was acccxnpanied by vacuolation andstarch mobilization like that in monospore germination. The paper also discusses the relations between monospores and separated and cultured cells.     

The influence of Fe^2＋ on growth and development of cells enzymatically isolated from Porphyra yezoensis blades

Fe^2＋ acted as an accessorial factor for many cellular enzymatic reactions is very important for seaweed growth and development, but the Fe^2＋ requirement in nori had not been seen. Porphyra yezoensis cells were separated enzymatically and cultured in a series of sterilized seawater media containing various concentrations of Fe^2＋. The growth development and cell were investigated in this work. Through this experiment, two biologically-meant concentration scales were found, one is low concentrations, 12.1-102.1μg/L, 10-100 times than that in seawater, favoring the development of isolated cells of Porphyra and the other was high concentrations, more than 10mg/L inhibiting the cell growth, leading to the deformity and shrinkage of the cells. At the concentration of 50 mg/L, the cells stopped growing and died eventually.     

Effects of Basic Fibroblast Growth Factor and Insulin-like Growth Factor on Cultured Cartilage Cells from Skate Raja porasa

Effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor Ⅱ(IGF-Ⅱ) on cartilage cells from proboscis of skate, Raja porasa Glinther, were investigated in this study. The cartilage cells were cultured in 20% FBS-supplemented MEM medium at 24℃. Twelve hours after culture initiation, the cartilage cells were treated with bFGF and IGF-Ⅱ at different concentration combinations. It was found that 20 ng/ml of bFGF or 80 ng/ml of IGF-Ⅱ was enough to have obvious stimulating effect on the growth and division of skate cartilage cells. Test of bFGF and IGF-Ⅱ together, revealed that 20 ng/ml of bFGF and 80 ng/ml of IGF-Ⅱ together had the best stimulating effect on the growth and division of skate cartilage cells. The cartilage cells cultured could form a monolayer at day 7.     

Enhancing the culturability of bacteria from the gastrointestinal tract of farmed adult turbot Scophthalmus maximus

Eighteen agar media were tested for the culture of gut-associated bacteria from farmed adult turbot(Scophthalmus maximus),including 16 agar media with or without 1% gastrointestinal(GI)supernatant,or with 2% or 4% GI supernatant.A total of 1 711 colonies were analyzed and 24 operational taxonomic units(OTUs)were identifi ed.The greatest bacterial diversity was isolated on Zobell 2216E/Zobell 2216E+ agar media,whereas MRS/MRS+ agar media produced a low diversity of colonies.Agar media with GI supernatant(1%,2%,or 4%)showed increased diversity and yielded different profi les of OTUs from the corresponding original media,suggesting that GI supernatant provides substances that enhance the culture effi ciency of bacteria from the turbot GI tract.The large majority of the colonies(82%)were γ-Proteobacteria,whereas 15.6% and 2.4% of colonies were Firmicutes and Actinobacteria,respectively.At the genus level,49.4% of all colonies were assigned to Vibrio.Other potential pathogens,including Pseudomonas,Photobacterium,and Enterobacter,and potential probiotics,including Bacillus,Paenibacillus,and Pseudomonas,were also isolated on agar media.Most cultured bacteria belonged to species that were fi rst described in the turbot GI tract.The impact of these species on turbot physiology and health should be investigated further.     

ISOLATION AND CULTIVATION OF THE VEGETATIVE CELLS OF PORPHYRA HAITANENSIS

This research deals mainly with the use of the vegetative cells of a marine red alga Porphyra hailanensis (laver) as seeds and their culture into young thalli for further cultivation in the sea. The experimental process was as follows. Enzymatically isolated vegetative cells were attached to rope substrates and cultured in the laboratory for a month until they became about 0.2-0.5 cm long sporelings and were then attached (7-15 sporelings per cm of rope) to nets for removal to the open sea for cultivation. A month after culturing, the thalli reached a maximum length of 50 cm (average of 20-30 cm). The thalli grew faster as the water temperature dropped from 21℃ to 17℃. It was proved that the vegetative cells isolated from a small thallus about 5 cm long could grow up into normal thalli after being kept frozen for a year. The results of this study show that vegetative cells can be used as new seeds to simplify the production of laver sporelings. This technique is a significant advance in the field of P     

STUDY ON THE RELATIONSHIP BETWEEN SPECIATION OF HEAVY METALS AND THEIR ECOTOXICITY——I. TOXICITY OF Cu, Cd , Pb AND Zn IN SEAWATER TO THREE MARINE ALGAE IN THE PRESENCE OF DIFFERENT COMPLEXATION AGENTS

Heavy metal is a main pollutant in the marine ecosystem . so study on the effect of heavy metal on phytoplankton is important . Algae (Chaetoceros sp . , Dunaliella sp . ., Dicrateria zhanjiangenis Hu . var . sp .) were laboratory cultured to observe the effect of heavy metals on their growth . The effect of different metal ion concentration , the detoxication effect of complexation agents and the growth of algae in different media and different nutrition levels were studied to evaluate the effect of metal speciation . It is proved that trace amount of heavy metals can stimulate the growth of algae cells but that high concentration is lethal . The sequence of toxicity is Cd2+>Zn2+>Pb2+ . In ordinary nutrition conditions , the detoxication sequence of complexation agents to Chaetoceros sp . is EDTA > sodium salicylate > sodium oxalate > sodium citrate > sulfanilic acid > O-phenanthroline . This is in good conformity with the stability constant sequence of these agents with copper and good evidence that t     

Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker.It can control the phleomycin resistance,and significantly increase the tolerance of hosts to zeocin.The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin.We selected ble as the selective marker for the genetic transformation of N.oculata.After the algal cells at a density of 2×10~7 cells mL~(-1) was digested with 4% hemicellulase and 2% driselase for 1 h,the protoplasts accounted for 90% of the total.The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii,yielding a recombinant expression construct p MS188.The construct was transferred into the protoplasts through electroporation(1kV,15 μS).The transformed protoplasts were cultured in fresh f/2 liquid medium,and selected on solid f/2 medium supplemented with 500 ngmL~(-1) zeocin.The PCR result proved that ble existed in the transformants.Three transformants had been cultured for at least 5 generations without losing ble.Southern blotting analysis showed that the ble has been integrated into the genome of N.oculata.The ble will serve as a new dominant selective marker in genetic engineering N.oculata.     

The influence of Fe~(2+) on growth and development of cells enzy-matically isolated from Porphyra yezoensis blades

1 INTRODUCTION Porphyra (also called nori in Japanese) is one of the most important algae in aquaculture in the world. In the early 1980’s, basic studies on cell and protoplast isolation, cultivation and “seedling” breeding of Porphyra were conducted in laboratory. Zhao and Zhang (1981) isolated and cultured isolated cells by grinding the nori thallus, and Lu (1983) isolated cells by digesting bacteria. An enzyme isolated from a sea snail, was used to isolate cells and protoplasts (T…     

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis.A new Sertoli cell line(POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages.Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P.olivaceus.Cells were optimally maintained at 25℃ in DMEM/F12 medium supplemented with fetal bovine serum,basic fibroblast growth factor,and epidermal growth factor.The growth curve of POSC showed a typical "S" shape.Chromosome analysis revealed that the cell line possessed the normal P.olivaceus diploid karyotype of 2n=48t.POSC expressed dmrt1 but not vasa,which was detected using RT-PCR and sequencing.Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL.Therefore,POSC appeared to consist of testicular Sertoli cells.Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid,with the transfection efficiency reaching 10%.This research not only offers an ideal model for further gene expression and regulation studies on P.olivaceus,but also serves as valuable material in studying fish spermatogenesis,Sertoli cell-germ cell interactions,and the mechanism of growth and development of testis.     

Seasonal variations of group-specific phytoplankton cell death in Xiamen Bay,China

The importance of phytoplankton cell death is being increasingly recognized,however,there are still no published reports on this in Xiamen Bay.In this study,the proportion of dead phytoplankton cells associated with environmental factors was assessed at a station in Xiamen Bay from December2012 to December 2013,using a cell digestion assay,which is an effective method to analyze dead/living cells in complex natural phytoplankton communities.The percentages of dead cells(%DC) in the total phytoplankton in summer(16%±6%) were lower than those in winter(27%±16%).Six groups of phytoplankton(G1-G6) were categorized by flow cytometry.These phytoplankton communities with diverse seasonal variations in%DC had different responses to environmental constraints.The main factors affecting mortality were temperature and salinity,while nutrient concentration showed little influence on phytoplankton death.Additionally,our results provide evidence that chlorophyll a concentrations had an inverse relationship with total phytoplankton%DC and viable cell abundance was more meaningful than total cells to explain variations in environmental parameters(such as Chl a).Moreover,the lowest mean%DC in total phytoplankton was 16%±6%at our sample site,which is in a subtropical area with high water temperatures,full solar radiation,and rich nutrients.This indicates that phytoplankton cell death is a process that cannot be ignored.In summary,phytoplankton cell death is important in understanding the dynamics of phytoplankton communities and the functioning of subtropical ecosystems.     

Ultrastructure of Single Cells, Callus-like and Monospore-like Cells in Porphyra yezoensis Ueda on Semi-solid Culture Medium

ＩＮＴＲＯＤＵＣＴＩＯＮＴｈｅｓｉｎｇｌｅｃｅｌｌｓａｎｄｐｒｏｔｏｐｌａｓｔｓｏｆＰｏｒｐｈｙｒａｇａｍｅｔｏｐｈｙｔｅｂｌａｄｅｓｃｏｕｌｄｄｅｖｅｌｏｐｉｎｔｏｐｌａｎｔｌｅｔｓｄｉ ｒｅｃｔｌｙｏｒｉｎｔｏｃａｌｌｕｓ ｌｉｋｅｃｅｌｌｓ (ＺｈａｏａｎｄＺｈａｎｇ ,1 981 ;Ｐｏｌｎｅ Ｆｕｌｌｅｒｅｔａｌ.,1 984 ;Ｃｈｅｎ ,1 989;ＷａｎｇａｎｄＹａｎ ,1 990 ;Ｇａｌｌｅｔａｌ.,1 993;Ｃｈｅｎ ,1 997;ＭｅｉａｎｄＦｅｉ,2 0 0 1 ) ,ａｎｄｔｈｅｉｒｇｒｏｗｔｈａｎｄｄｅｖｅｌｏｐｍｅｎｔｕｎｄｅｒ…     

Ultrastructure of Single Cells, Callus-like and Monosore-like Cells in Porphyra yezoensis Ueda on Semi solid Culture Medium

It had been demonstrated that individual cells or protoplasts isolated from Porphyra thallus by enzyme could develop into normal leafy thalli in the same way as monospores, and that isolated cells develop in different way in liquid and on semi solid media. The authors observed the ultrastructure of isolated vegetative cells cultured on semi solid media and compared them with those of monospores and isolated cells cultured in liquid media. The results showed that subcellular structures were quite different among cells in different conditions. In their development, isolated cells on semi solid media did not show the characteristic subcellular feature of monospore formation, such as production of fibrous vesicles. Callus like cells formed on semi solid media underwent a distinctive modification in cellular organization. They developed characteristic cell inclusions and a special 2 layer cell covering. Golgi bodies, ER, starch grains, mitochondria. Vacuoles were not commonly found in them.     

The ultrastructure of separated and cultured cell of Porphyra yezoensis

There are many reports that cells (protoplasts) separated from the thallus ofPorphyra by enzyme can develop to normal leafy thalli in the same way as monospores. But there are few investigations on the subcellular structure of the isolated vegetative cell for comparison with the subcellular structure of monospores. To clarify whether the separated and cultured cells undergo the same or similar ultrastructure changes during culture and germination as monospores undergo in their formation and germination, we observed their ultrastructure, compared them with those of the monospore and found that the ultrastructure of separated and cultured cells did not have the characteristic feature as that of monospore formation, such as production of small and large fibrous vesicles, but was accompanied by vacuolation and starch mobilization like that in monospore germination. The paper also discusses the relations between monospores and separated and cultured cells. Contribution No. 3875 from the Institute of Oceanology, Chinese Academy of Sciences. Project 39770593 supported by NSFC; Project 96-C01-05-01 of the 9th Five-Year Plan supported by Science and Technology Commision of China.     

Study on monospore-like cells fromPorphyra haitanensis

Porphyra haitanensis andP. yezoensis are two mainPorphyra species cultured in China. Their life histories are slightly different. So far we have not observed thatP. haitanensis naturally produces monospores developing into thalli.P. yezoensis produces monospores which directly germinate into young thalli used in cultivation (Zeng, et al., 1985). Some somatic cells inP. yezoensis develop in vitro into monospore-like cells which later grow into young thalli (Lu, 1983). Studies on whether or not somatic cells inP. haitanensis can produce in vitro monospore-like cells that later grow into young thalli is important for understanding its life history and for culturing new varieties.     

Initiation of primary cell culture from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri tsingtauense</Emphasis>

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C. Supported by Doctoral Initial Fund of Ludong University (No.43304)     

Isolation and cultivation of the vegetative cells ofPorphyra haitanensis

This research deals mainly with the use of the vegetative cells of a marine red algaPorphyra haitanensis (laver) as seeds and their culture into young thalli for further cultivation in the sea. The experimental process was as follows. Enzymatically isolated vegetative cells were attached to rope substrates and cultured in the laboratory for a month until they became about 0.2–0.5 cm long sporelings and were then attached (7–15 sporelings per cm of rope) to nets for removal to the open sea for cultivation. A month after culturing, the thalli reached a maximum length of 50 cm (average of 20–30 cm). The thalli grew faster as the water temperature dropped from 21°C to 17°C. It was proved that the vegetative cells isolated from a small thallus about 5 cm long could grow up into normal thalli after being kept frozen for a year. The results of this study show that vegetative cells can be used as new seeds to simplify the production of laver sporelings. This technique is a significant advance in the field ofPorphyra culture.     

Toxic effects of Karenia mikimotoi extracts on mammalian cells

Karenia is one of the most harmful and representative red tide genus in a temperate zone. Blooms caused by this genus have resulted in massive fish death in the South China Sea and the East China Sea. However, the potential effects of this dinoflagellate on human health through the transfer of toxins via marine food webs, and the mechanisms of toxicity, are still unknown. Therefore, we examined the toxic effects of a strain of K. mikimotoi (isolated from the South China Sea) on the proliferation and morphology of four mammalian cell lines (two normal cell lines and two cancer cell lines). In addition, we carried out a preliminary investigation on the mechanism of toxicity of the alga. The results show that the polar lipid-soluble component of K. mikimotoi significantly inhibited proliferation of the four cell lines, and resulted in the cells becoming spherical, swollen and damaged. The result of Annexin V and PI double-staining confirmed that cell membranes were disrupted. The malonaldehyde (MDA) contents in the medium of the four cell lines treated with the polar-lipid extracts all increased significantly, which indicates that the polar-lipid toxins produced by K. mikimotoi could adversely affect mammalian cells by inducing lipid peroxidation. We conclude that K. mikimotoi is a potential threat to human health, and the comprehensive effect of this dinoflagellate and its mechanisms should be investigated further.     

Early stage differentiation of thallus cells ofPorphyra haitanensis (Rhodophyta)

The early stage differentiation of thallus cells ofPorphyra haitanensis T. J. Chang et B. F. Zheng was studied. Protoplasts or single cells were isolated from the blades using enzyme mixture comprising 2% sea snail gut enzyme and 1% cellulase. The isolated protoplasts or single cells were incubated in the MES medium. The cell differentiations were examined under the microscope at intervals after incubation. Four types of cell differentiation, namely, normal, abnormal, carposporangial and spermatorangial, and rhizoidal types, were observed. Since normal cell differentiations occur mostly in small thalli 50 mm in length and middle portions of big thalli 200 mm in length, it is essential to select tissues from these two kinds of thalli essential for commercial production.