• Research in Plant Disease
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• v.15 no.1
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• pp.46-50
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• 2009

Cytochalasin E (CE) is a secondary metabolite secreted by Rosellinia necatrix, caused by white root rot, and has toxicity to apple as a toxin during disease progress. This study was conducted to demonstrate the relationship between the production of CE and its pathogenicity. CE producing isolates and non-producing isolates of R. nectatrix were isolated from the mycerial mat of diseased roots and was detected on that using a TLC and HPLC analysis and in vivo pathogenicity test. CE non-producing isolates were not pathogenic to apple roots and not detected CE by TLC and HPLC analysis. It was shown that the production of CE was related to the pathogenicity of R. nectatrix.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.43-49
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• 1997

The objective of this study is to determine developmental pattern and cell allocation to the ICM and TE in haploid and diploid of embryos following parthenogenetic activation and in vitro fertilization. The incidence of development to blastocyst was lower in the combined treatments of ethanol stimulation and cytochalasin B as compared to the control. However, the combined ethanol stimulation and cytochalasin B treatment (diploid) accelerated development to the blastocyst as compared to the ethanol treatment alone (haploid). Significantly reduction in the average number of total cells and ICM was observed in the parthenotes alone as compared to fetilized embryos, but those of combined ethanol stimulation and cytochalasin B treatment embryos were significantly increased as compared to ethanol alone embryos. These results suggested that the ploidy affects preimplantation developmental patters and cell allocation to the ICM and TE in the porcine.

• Biomedical Science Letters
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• v.14 no.3
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• pp.179-186
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• 2008

Matrix metalloproteinases (MMPs), also designated matrixins, hydrolyze components of the extracellular matrix. These proteinases playa central role in many biological processes, such as embryogenesis, normal tissue remodeling, wound healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue ulceration. In previous data, disruption of the actin cytoskeleton by cytochalasin D (CD) inhibited NO-induced apoptosis, dedifferentiation, cyclooxygenase (COX)-2 expression, and prostaglandin $E_2$ production in chondrocytes cultured on plastic or during cartilage explants culture. In this study, we investigated the effects of the actin cytoskeleton architecture on MMP-2 expression and dedifferentiation by CD in rabbit articular chondrocytes. Rabbit articular chondrocytes were prepared from cartilage slices of 2-weeks-old New Zealand white rabbits by enzymatic digestion. CD was used as a disruptor of actin cytoskeleton. In this experiments measuring CD dose response, primary chondrocytes were treated with various concentrations of CD for 24h. The actin disruption was determined by immunostaining. MMP-2 expression levels were determined by immunoblot analysis and Reverse transcriptase-Polymerase chain reaction (RT-PCR) and MMP-2 activity was determined by gelatin zymography. We found that cell morphological change and up-regulation of MMP-2 expression by CD as determined via immunostaining, gelatin zymography and immunoblotting. Moreover, CD induced MMP-2 transcription was detected by RT-PCR. Also, CD-induced type II collagen expression was inhibited by MMP-2 inhibitor I treatment. Our results indicate that CD up-regulated MMP-2 activation causes dedifferentiation of articular chondrocyte.

• Biomedical Science Letters
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• v.15 no.4
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• pp.343-347
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• 2009

Cytochalasin D (CD) is known as a disruptor of actin cytoskeleton architecture in chondrocytes. We have studied the role of CD in retinoic acid (RA) caused dedifferentiation and inflammation responses in rabbit articular chondrocytes. We have examined the effect of CD on RA induced dedifferentiation of chondrocytes. CD inhibited RA induced dedifferentiation determined by Western blot analysis and Alcian blue staining in rabbit articular chondrocytes. Also, CD additionally reduced inflammation response molecules such as cyclooxygenase-2 (COX-2) and prostaglandin $E_2$ ($PGE_2$) in RA treated cells. Treatment of CD reduced phosphorylation of p38 by treatment of RA. Inhibiton of p38kinase with SB203580 reduced expression of COX-2 and production of $PGE_2$ by treatment of CD in RA treated cells. But, Inhibiton of p38kinase with SB203580 did not any relationship with effect of CD on RA caused dedifferentiation. In summary, our results indicate that CD regulates RA reduced expression of COX-2 and production of PGE2 via p38kinase pathway.

• International Journal of Oral Biology
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• v.35 no.2
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• pp.43-49
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• 2010

Enterococcus faecalis, a gram-positive bacterium, has been implicated in endodontic infections, particularly in chronic apical periodontitis. Proinflammatory cytokines, including tumor necrosis factor-$\alpha$ (TNF-$\alpha$), are involved in the pathogenesis of these apical lesions. E. faecalis has been reported to stimulate macrophages to produce TNF-$\alpha$. The present study investigated the mechanisms involved in TNF-$\alpha$ production by a murine macrophage cell line, RAW 264.7 in response to exposure to E. faecalis. Both live and heat-killed E. faecalis induced high levels of gene expression and protein release of TNF-$\alpha$. Treatment of RAW 264.7 cells with cytochalasin D, an inhibitor of endocytosis, prevented the mRNA up-regulation of TNF-$\alpha$ by E. faecalis. In addition, antioxidant treatment reduced TNF-$\alpha$ production to baseline levels. Inhibition of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase also significantly attenuated E. faecalis-induced TNF-$\alpha$ expression by RAW 264.7 cells. Furthermore, activation of NF-${\kappa}B$ and AP-1 in RAW 264.7 cells was also stimulated by E. faecalis. These results suggest that the phagocytic uptake of bacteria is necessary for the induction of TNF-$\alpha$ in E. faecalis-stimulated macrophages, and that the underlying intracellular signaling pathways involve reactive oxygen species, ERK, p38 MAP kinase, NF-${\kappa}B$, and AP-1.

• The korean journal of orthodontics
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• v.28 no.6 s.71
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• pp.915-926
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• 1998

The cytoskeleton has been shown to form a network, connecting the extracelluar matrix via integrin with the nucleus and the cytoplasmic constituents of the cell. It is therefore assumed that the cytoskeleton may mediate signals generated by perturbations originating in the matrix. The purpose of this study is to examine the effect of cytoskeletal change on osteoblastic cell activities. The author cultured osteoblastic cells obtained from neonatal mouse calvaria. The cells were teated with cytochalasin B(CB) or colchicine (COL) at four concentrations for 3 hours and after another 24 hours the conditioned media was collected and assayed for prostaglandin $E_2\;(PGE_2)$, interleukin-6(IL-6), tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and matrix metalloproteinase-1(MMP-1). In addition, the cytoskeletal protein actin were observed by immuno-fluorescence. The results were as follows: 1. The production of $PGE_2$ showed the tendency to be increased in CB-treated group. $PGE_2$ was increased in COL-treated group dose-dependantly, 2. IL-6 production, in CB-treated group, was increased, except at 1.0 ${\mu}g/ml$. IL-6 was induced in COL-treated group. 3. TNF-$\alpha$ production was increased in CB-treated group, except at 1.0 ${\mu}g/ml$, and in COL-treated group, that was increased. 4. The MMP-1 production was decreased in CB-treated soup and was not changed in COL-treated group, which could be selectively visualized by immunoblotting with monospecific antibody. 5. The cytoskeletal actin stress fibers were disappeared and the cells showed to be rounded in CB-treated group. These results indicated that there are a relationship between the cytoskeletal rearrangements and osteoblastic cell activities, especially in release of paracrine/autocrine factors, such as $PGE_2$, IL-6, and TNF-$\alpha$.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.245-252
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• 2002

This study was to evaluate the in vitro survival of bovine enucleated MII (eMII) oocytes according to minimum volume cooling (MVC) freezing method and activation timing, and their in vitro development after somatic cell nuclear transfer (SONT). in vitro matured bovine oocytes for 20 h were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst, and their 1st polar body and MII plate were removed by enucleation micropipette under UV filter. Also, eMII oocytes were subjected to activation after (group II) and before (group III) vitrification in 5 ${\mu}{\textrm}{m}$ ionomycin added CRlaa medium for 5 min. For vitrification, eMll oocytes were pretreated with EG10 for 5 min, exposed to EG30 for 30 sec and then directly plunged into L$N_2$. Thawing was taken by 4-step procedures at 37$^{\circ}C$. Survived eMII oocytes were subjected to SONT with cultured adult bovine ear cells. Reconstructed oocytes were cultured in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide and 2.5 $\mu\textrm{g}$/$m\ell$ of cytochalasin D added CRlaa medium for 1 h, and then in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide added CRlaa medium for 4 h. Subsequently, the reconstructed oocytes were incubated for 2 days and cleaved embryos were further cultured on cumulus-cell monolayer drop in CRlaa medium for 6 days. Survival rates of bovine vitrified-thawed eMII oocytes in group II (activation after vitrification and thawing) and III (activation before vitrification) were 81.0% and 84.9%, respectively. Fusion rates of cytoplasts and oocytes in group II and III were 69.0% and 70.0%, respectively, and their results were not different with non-frozen NT group (control, 75.2%). Although their cleaved rates (53.4% and 58.4%) were not different, cytoplasmic fragment rate in group II (32.8%) was significantly higher than that in group III (15.6%)(P<0.05). Also, subsequent development rate into >morula in group II (8.6%) was low than that in group III(15.6%). However, in vitro development rate in group III was not different with that in control (24.8%). This result suggested that MVC method was appropriate freezing method for the bovine eMII oocytes and vitrified eMII oocytes after pre-activation could support in vitro embryonic development after SONT as equally well as fresh oocytes.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.1-7
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• 2010

The objective of this study was to examine the effect of vitamin B (pantothenic acid, folic acid, and myo-inositol) that was supplemented to embryo culture medium on in vitro development of parthenogenetically activated (PA) pig embryos. Cumulus-oocyte complexes derived from slaughtered ovaries were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (hCG and eCG) for the first 22 h and then further cultured in hormone-free medium for an additional 22 h. After maturation culture, metaphase II oocytes that extruded 1st polar body were electrically activated and treated with $5.0\;{\mu}g/ml$ cytochalasin B for 4 h. Then, PA embryos were cultured for 7 days in a modified NCSU-23 that was supplemented with pantothenic acid, myo-inositol, or folic acid at different concentrations ($3{\sim}300\;{\mu}M$) according to the experimental design. Myo-inositol added to culture medium did not show any beneficial or inhibitory effects on embryo cleavage and blastocyst formation. However, $300\;{\mu}M$ pantothenic acid significantly inhibited blastocyst formation compared to control (no addition) (24% vs. 36%, p<0.05). Folic acid ($300\;{\mu}M$) significantly (p<0.05) increased blastocyst formation (56%) compared to control (41%). Our results demonstrated that in vitro development of PA embryos was significantly influenced by vitamin B and addition of $300\;{\mu}M$ folic acid to culture medium improved in vitro development of pig PA embryos.

• Environmental Analysis Health and Toxicology
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• v.28
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• pp.3.1-3.8
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• 2013

Objectives We investigated the genotoxic effects of 40-59 nm silver nanoparticles (Ag-NPs) by bacterial reverse mutation assay (Ames test), in vitro comet assay and micronucleus (MN) assay. In particular, we directly compared the effect of cytochalasin B (cytoB) and rat liver homogenate (S9 mix) in the formation of MN by Ag-NPs. Methods Before testing, we confirmed that Ag-NPs were completely dispersed in the experimental medium by sonication (three times in 1 minute) and filtration ($0.2{\mu}m$ pore size filter), and then we measured their size in a zeta potential analyzer. After that the genotoxicity were measured and especially, S9 mix and with and without cytoB were compared one another in MN assay. Results Ames test using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains revealed that Ag-NPs with or without S9 mix did not display a mutagenic effect. The genotoxicity of Ag-NPs was also evaluated in a mammalian cell system using Chinese hamster ovary cells. The results revealed that Ag-NPs stimulated DNA breakage and MN formation with or without S9 mix in a dose-dependent manner (from $0.01{\mu}g/mL$ to $10{\mu}g/mL$). In particular, MN induction was affected by cytoB. Conclusions All of our findings, with the exception of the Ames test results, indicate that Ag-NPs show genotoxic effects in mammalian cell system. In addition, present study suggests the potential error due to use of cytoB in genotoxic test of nanoparticles.

• Journal of Life Science
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• v.18 no.7
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• pp.952-957
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• 2008

We evaluated the effects of various factors (e.g., serum, inhibitors of protein synthesis, and cytoskeleton and protein kinases) on early PMA-induced THP-1 cell adhesion using an adhesion assay with Sulforhodamine B (SRB) staining, which was used to assess the proliferation of the attached cells. THP-1 cell adhesion to a plastic substrate was detected 1 hr after exposure to Phorbol 12-Myristate 13-Acetate (PMA) and peaked after 18 hr. At concentrations > 25 nM PMA, the level of adhesion did not change. Based on our preliminary results, we used 25 nM PMA and 5 hr of culture as standard assay conditions. Early PMA-induced cell adhesion was not affected by the presence of serum or PD 98059 in the culture medium, but was affected by the addition of PKC inhibitors and cycloheximide. In the presence of actin inhibitor with PMA, the cell adhesion increased when comparing with PMA treatment only. Thus, early PMA-induced adhesion of THP-1 cells does not require serum in the culture medium, MAP-kinase activation, or actin polymerization, but does require de novo protein synthesis and PKC activation. Our SRB-based cell adhesion assay may be used to screen other PKC inhibitors.