• Molecules and Cells
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• v.39 no.10
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• pp.768-775
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• 2016

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

• Journal of the Korean institute of surface engineering
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• v.17 no.2
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• pp.29-40
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• 1984

The structure of rapidly solidified Al-Si-based alloys and its relationship to subsequent anodic film growth in near neutral and acid solutions have been investigated. Solidification of the alloys proceeds via pre-dendritic nuclei, associated with rugosity of the casting surface, from which cellular-type growth, comprised of aluminium-rich material surrounded by silicon-containing material, emanates. Observation of ultramicrotomed sections of the alloys and their anodic films reveals the local oxidation of the silicon-rich phase and its incorporation into the anodic alumina film, formed in near neutral solutions. Such incorporation occurs but resultant isolation of the silicon-rich phase is not possible for anodizing in phosphoric acid, and a three-dimensional network of the oxidized silicon-containing phase, with continuing development of porous anodic alumina, is observed.

• Korean Journal of Veterinary Research
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• v.51 no.3
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• pp.209-216
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• 2011

Four strains of fowl adenovirus (FAdV) were isolated from 4 flocks of broiler or layer chickens affected by hydropericardium syndrome in Korea. These FAdVs were classified as serotype 4 by restriction fragment length polymorphism patterns of hexon genes and whole genomes. The virus exhibited cytopathic effects consisting of rounding, ballooning and clustering in primary chicken embryo liver cell cultures. In transmission electron microscopy, virus particles in hexagonal shape aggregated exclusively in the nuclei of hepatocytes of the chickens as the typical appearances in adenovirus infections. Buoyant density of the virus in cesium chloride (CsCl) was 1.34 g/mL. The virus was stable to chloroform, ether, 50~70% ethanol, acidic condition at pH 3, 0.25% trypsin (1 : 250), heat at $50^{\circ}C$ for 30 min, but labile to 100% ethanol, heat at $52{\sim}60^{\circ}C$ for 30 min, 1 M $MgCl_2$ at $50^{\circ}C$ for 1 h, 1 : 2,000 formalin (37%). All of the physicochemical properties pertained to the characteristics of adenoviruses. Eight viral polypeptides were determined in CsCl-purified virus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

• KSBB Journal
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• v.5 no.3
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• pp.255-261
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• 1990

Cell fusion between complementary mutants isolated from astaxanthin-producing yeast, Phaffia rhodozyma, was carried out to obtain astaxanthin-overproducing strains by protoplast fusion technique. The frequency of protoplast fusion was ranged from 2.3$\times$10-5 to 6.0$\times$10-5, and nuclear fusion in the cells of hybrids was demonstrated by several techniques such as isolation of recombinants after mitotic segregation of parental genetic markers, estimation of DNA content, direct observation of nuclei with nuclear staining, and comparison of survival rate to UV exposure. One of several hybrids, Fl, showed approximately 3-fold increase in astaxanthin content when compared with wild parent.

• Molecules and Cells
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• v.27 no.4
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• pp.443-447
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• 2009

This paper reports on the structural rearrangement of satellite DNA type I repeats and heterochromatin during the dedifferentiation and cell cycling of mesophyll protoplasts of cucumber (Cucumis sativus). These repeats were localized in the telomeric heterochromatin of cucumber chromosomes and in the chromocenters of interphase nuclei. The dramatic reduction of heterochromatin involves decondensation of subtelomeric repeats in freshly isolated protoplasts; however, there are not a great many remarkable changes in the expression profile. In spite of that, reformation of the chromocenters, occurring 48 h after protoplast isolation, is accompanied by recondensation of satellite DNA type I; however, only partial reassembly of these repeats was revealed. In this study, FISH and a flow cytometry assay show a correlation between the partial chromocenter and the repeats reassembly, and with the reentry of cultivated protoplasts into the cell cycle and first cell division. After that, divided cells displayed a higher variability in the expression profile than did leaves' mesophyll cells and protoplasts.

• Korean Journal of Veterinary Research
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• v.36 no.4
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• pp.845-858
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• 1996

The purpose of this study was to establish early diagnostic methods for the detection of Aujeszky's disease viral antigens and nucleic acid in nasal cells, and buffy coats from experimentally infected living pigs by a combination of immunocytochemistry, in situ hybridization with digoxigenin(DIG)-labled probe and electron microscopy. Forty days old piglets were inoculated intranasally with $10^{7.0}TCID_{50}$ of Aujeszky's disease virus (ADV, NYJ-1-87 strain). The viral antigens and nucleic acid of ADV were detected in nasal cells, and buffy coat for 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopical method. The results were compared with conventional methods such as a porcine Aujeszky's disease serodiagnostic(PAD) kit, neutralization test(NT) and virus isolation. 1. The viral antigens, nucleic acids and capsids of ADV were detected in nasal cells, buffy coats from 3 days to 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopy, respectively. 2. When viral antigens were detected by the immunocytochemical technique, a diffuse brown deposit was observed in the nucleus and cytoplasm of nasal cells, buffy coats and PK-15 cells under a microscope. 3. DIG-labeled DNA probe was prepared by amplification of conserved sequence of recombinant ADV-gp50 clone with polymerase chain reacction. When ADV-DNA was detected by ISH with DIG-labeled probe, purplish blue pigmentation were observed in the nuclei and cytoplasms of ADV-infected cells under a microscope. Positive signals were observed in nasal cells and in the buffy coat and PK-15 cells at the first day after inoculation. 4. Where ADV-capsids were detected by transmission electron microscopical method, aggregation of capsids was observed in the nuclei and cytoplasms of nasal cells, buffy coats and PK-15 cells. The results suggested that these methods were considered as the highly sensitive and reliable tools for rapid and confirmative diagnosis of Aujeszky's disease in living pigs.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.290-296
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• 1998

Interspecific hybrids between Aspergillus oryzae var. oryzae and Penicillium chrysogenum (Tyr$\^$-/), high alkaline protease producing fungi, were obtained by nuclear transfer technique. Nuclei isolated from the wild type Aspergillus oryzae var. oryzae strain were transferred into auxotrophic Penicillium chrysogenum mutants and selected the new strains showing an increased protein degrading capability. Maximum production of protoplasts were obtained by 1% Novozym 234 at $30^{\circ}C$ for 3 hours and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6M KCl. Frequencies of hybrid formation by nuclear transfer were 1.3${\times}$10$\^$-3/∼2.8${\times}$10$\^$-3/. They could be suggested as an aneuploid by the observation of genetic stability, conidial size, DNA content, and nuclear strain. The hybrids showed 1.1～2.2 fold higher alkaline pretense activities than parental strains.

• The Korean Journal of Mycology
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• v.27 no.6 s.93
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• pp.399-405
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• 1999

Intergeneric hybrids between Saccharomyccopsis fiburigera KCTC 7393 and Saccharomyces cerevisiae KCTC 7049 (tyr-, ura-) were obtained by nuclear transfer technique. Nuclei isolated from the wild type S. fiburigera strain were transfered into auxotrophic S. cerevisiae mutants and new strains showing an increased starch degrading capability were selected. Maximum production of protoplasts was obtained from the treatment with 0.1 % Novozym 234 at $30^{\circ}C$ for 90 min, and most effective osmotic stabilizer for the isolation of protoplasts was 0.6 M KCl at pH 5.8. The frequency of protoplast regeneration was 14.64% under the conditions. Genectic stability, conidial size, DNA content, and nuclear stain suggested that the fusants were aneuploidy. The specific activity of ${\alpha}-amylase$ was observed to increase about $1.2{\sim}1.9$ folds.

• Journal of Life Science
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• v.21 no.12
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• pp.1666-1677
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• 2011

The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel gene, the $mas1^+$($\underline{mi}$tosis $\underline{as}$sociated protein) gene, a homolog of human CIP29/Hcc1, was isolated and characterized from fission yeast Schizosaccharomyces pombe (S. pombe) using a gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 245 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that a PCB ($\underline{p}$ombe cell $\underline{c}$ycle $\underline{b}$ox) is located in the promoter region, which controls M-$G_1$ specific transcription in S. pombe. The quantitative analysis of the $mas1^+$ transcript against $adh1^+$ showed that the pattern of expression is similar to that of the septation index. Cytokinesis of mas1 mutant was greatly delayed at $25^{\circ}C$ and $36^{\circ}C$, and a large number of multi-septate cells were produced. The mas1 mutant had 2C, 4C and 6C DNA contents, as determined by FACS analysis. In addition, the number of multi-septate cells significantly increased. When cells were cultured in nitrogen starvation medium to increase proliferation, the abnormal phenotypes of mas1 mutant dramatically increased. These phenotypes could be rescued by an overexpression of the $mas1^+$ gene. The mas1 protein localized in the nuclei of S. pombe and human HeLa cells, as evidenced by Mas1-EGFP signals. The abnormal growth pattern and the morphology of mas1 mutant were complemented by a plasmid carrying human CIP29/Hcc-1cDNA. In addition, CIP29 /Hcc-1 transcript level increased in active cell proliferation stages in the developing mouse embryos. These results indicate that the $mas1^+$ ishomologous to the human CIP29/Hcc1 gene and is involved in cytokinesis and cell shape control.

• Journal of Korean Society of Forest Science
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• v.21 no.1
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• pp.1-34
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• 1974

The author intended to investigate external and internal changes in the cone structure, changes in water content, sugar, fat and protein during the period of seed maturation which bears a proper germinability. The experimental results can be summarized as in the following. 1. Male flowers 1) Pollen-mother cells occur as a mass from late in April to early in May, and form pollen tetrads through meiosis early and middle of May. Pollen with simple nucleus reach maturity late in May. 2) Stamen number of a male flower is almost same as the scale number of cone and is 69-102 stamens. One stamen includes 5800-7300 pollen. 3) The shape is round and elliptical, both of a pollen has air-sac with $80-91{\mu}$ in length, and has cuticlar exine and cellulose intine. 4) Pollen germinate in 68 hours at $25^{\circ}C$ with distilled water of pH 6.0, 2% sugar and 0.8% agar. 2. Female flowers 1) Ovuliferous scales grow rapidly in late April, and differentiation of ovules begins early in May. Embryo-sac-mother cells produce pollen tetrads through meiosis in the middle of May, and flower in late May. 2) The pollinated female flowers show repeated divisions of embryo-sac nucleus, and a great number of free nuclei form a mass for overwintering. Morphogenesis of isolation in the mass structure takes place from the middle of March, and that forms albuminous bodies of aivealus in early May. 3. Formation of pollinators and embryos. 1) Archegonia produce archegonial initial cells in the middle and late April, and pollinators are produced in the late April and late in early May. 2) After pollination, Oespore nuclei are seen to divide in the late May forming a layer of suspensor from the diaphragm in early June and in the middle of June. Thus this happens to show 4 pro-embryos. The organ of embryos begins to differentiate 1 pro-embryo and reachs perfect maturation in late August. 4. The growth of cones 1) In the year of flowering, strobiles grow during the period from the middle of June to the middle of July, and do not grow after the middle of August. Strobiles grow 1.6 times more in length 3.3 times short in diameter and about 22 times more weight than those of female flower in the year of flowering. 2) The cones at the adult stage grow 7 times longer in diameter, 12-15 times shorter diameter than those of strobiles after flowering. 3) Cone has 96-133 scales with the ratio of scale to be 69-80% and the length of cone is 11-13cm. Diameter is 5-8cm with 160-190g weight, and the seed number of it is 90-150 having empty seed ratio of 8-15%. 5. Formation of seed-coats 1) The layers of outer seed-coat become most for the width of $703{\mu}$ in the middle of July. At the adult stage of seed, it becomes $550-580{\mu}$ in size by decreasing moisture content. Then a horny and the cortical tissue of outer coats become differentiated. 2) The outer seed-coat of mature seeds forms epidermal cells of 3-4 layers and the stone cells of 16-21 layers. The interior part of it becomes parenchyma layer of 1 or 2 rows. 3) Inner seed-coat is formed 2 months earlier than the outer seed-coat in the middle of May, having the most width of inner seed-coat $667{\mu}$. At the adult stage it loses to $80-90{\mu}$. 6. Change in moisture content After pollination moisture content becomes gradually increased at the top in the early June and becomes markedly decreased in the middle of August. At the adult stage it shows 43~48% in cone, 23~25% in the outer seed-coat, 32~37% in the inner seed-coat, 23~26% in the inner seed-coat and endosperm and embryo, 21~24% in the embryo and endosperm, 36~40% in the embryos. 7. The content compositions of seed 1) Fat contents become gradually increased after the early May, at the adult stage it occupies 65~85% more fat than walnut and palm. Embryo includes 78.8% fat, and 57.0% fat in endosperm. 2) Sugar content after pollination becomes greatly increased as in the case of reducing sugar, while non-reducing sugar becomes increased in the early June. 3) Crude protein content becomes gradually increased after the early May, and at the adult stage it becomes 48.8%. Endosperm is made up with more protein than embryo. 8. The test of germination The collected optimum period of Pinus koraiensis seeds at an adequate maturity was collected in the early September, and used for the germination test of reduction-method and embryo culture. Seeds were taken at the interval of 7 days from the middle of July to the middle of September for the germination test at germination apparatus.