• Animal cells and systems
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• v.7 no.3
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• pp.227-235
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• 2003

Bisphenol A (BPA), nonylphenol (NP), and 4-tert-octylphenol (OP) are known endocrine disrupting chemicals (EDCs) with estrogenic activity in fish. This study compared the effects of BPA, NP and OP on in vitro vitellogenin (VTG) synthesis in primary cultures of hepatocytes of the Chinese minnow Phoxinus oxycephalus. The VTG secreted into the culture medium was measured using enzyme-linked immunosorbent assay (ELISA), which we developed in this study using an antibody prepared from homogenates of Chinese minnow egg. VTG synthesis was induced by estradiol-17$\beta$ ($E_2$) and phenols (BPA, NP and OP) treatment. $E_2$ at concentrations of 10$^{-6}$ M or higher increased VTG levels significantly (P < 0.05). Exposure to 10^5\;M\;BPA\;or\;10^-4$M NP and OPinduced in vitro VTG synthesis (P < 0.01). However, $10^-3$ M BPA, NP or OP did not induce VTG synthesis. These results suggest that SPA has the highest estrogenic potential in Chinese minnow hepatocytes. Tamoxifen, an anti-estrogen, drastically blocked the production of VTG by phenols (BPA, NP and OP) suggesting that phenols (BPA, NP and OP) may act via binding to estrogen receptor (ER) in Chinese minnow hepatocytes.

• Animal cells and systems
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• v.16 no.2
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• pp.145-153
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• 2012

The pear psylla, $Cacopsylla$ $pyricola$ (Hemiptera: Psyllidae), is a serious insect pest of commercial pear crops. The species, which resides on pear trees throughout its life cycle, is rapidly spreading in some regions of the world. The population genetic structure of the species collected from several pear orchards in Korea was studied to understand the nature of dispersal and field ecology of the species. The 658-bp region of mitochondrial COI gene and the 716-bp long complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA were sequenced. Unlike other previously studied insect pests, the COI-based genetic diversity of the pear psylla was extremely low (maximum sequence divergence of 0.15%). This finding allowed us to conclude that the species may have been introduced in Korea relatively recently. ITS2 sequence-based analyses of phylogeny, population differentiation, gene flow, and hierarchical population structure all concordantly suggested that the pear psylla populations in Korea are neither genetically isolated nor hampered for gene flow. These genetic data are concordant with the dispersal of an overwintering winterform morph outside the non-pear habitat in the fall.

• Animal cells and systems
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• v.16 no.2
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• pp.162-171
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• 2012

We investigated food webs of a $Salix$ $subfragilis$-dominated wetland in the Janghang wetland in the Hangang estuary, which is very close to the Demilitarized Zone, along the west coast of Korea. Our study focused on understanding sesarmine crab ($Sesarma$ $dehaani$)-related food webs in a $S.$ $subfragilis$ forest. For our study, we used carbon and nitrogen stable isotopes and fatty acid biomarkers. We collected samples of plants, animals, and detrital sediment from four quadrats ($5{\times}5m^2$) set in the $S.$ $subfragilis$ community. Samples were collected from September 2006 to June 2009, except during the winter hibernation period of $S.$ $dehaani$. In the wet season, the sediment showed relatively high ${\delta}^{13}C$ and low ${\delta}^{15}N$ signatures compared with relatively low ${\delta}13C$ and high ${\delta}15N$ signatures in the dry season. Mature $S.$ $dehaani$ appeared to feed on fresh leaves and other carbon sources, such as immature individuals or fish, in addition to detrital sediment, which appeared to be the main carbon source for immature crabs. Principal component analysis of fatty acid biomarkers of $S.$ $dehaani$ showed a clear difference between immature individuals (10-30 mm) and mature ones (larger than 30 mm), indicating that the main food source for immature crabs was detrital sediment, whereas mature crabs foraged plants in addition to consuming detrital sediment. On the basis of our results from stable isotope and fatty acid analyses, mature $S.$ $dehaani$ appeared to feed on detrital sediment and fresh leaves of $S.$ $subfragilis$ in summer in addition to engaging in cannibalism of immature individuals.

• Animal cells and systems
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• v.16 no.2
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• pp.104-113
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• 2012

The nucleolus is a distinct nuclear territory involved in the compartmentalization of nuclear functions. There is some evidence of a relationship between nuclear fragmentation during spermatogenesis and chromatoid body (CB) formation. The CB is a typical cytoplasmic organelle of haploid germ cells, and is involved in RNA and protein accumulation for later germ-cell differentiation. The goal of this study was to qualitatively and quantitatively describe the nucleolar cycle during the spermatogenesis of $Phrynops$ $geoffroanus$ (Reptilia Testudines), and compare this nucleolar fragmentation with CB formation in this species through the use of cytochemical and ultrastructural analysis. Qualitative analysis showed a fragmentation of the nuclear material after pachytene of the first meiotic division in the primary spermatocytes. Quantitative analysis of the nucleolar cycle revealed a significant difference in the number of nucleoli and in the size of the nucleolus between spermatogonia and early spermatids. Using ultrastructural analysis, we recorded the beginning of the CB formation process in the cytoplasm of primary spermatocytes at the same time as when nuclear fragmentation occurs. In the cytoplasm of primary spermatocytes, the CB was observed in association with mitochondrial aggregates and the Golgi complex. In the cytoplasm of early spermatids, the CB was observed in association with lipid droplets. In conclusion, our data show that the nucleolus plays a role in the CB formation process. During spermatogenesis of $P.$ $geoffroanus$, the CB is involved in some important biological processes, including acrosome formation and mitochondrial migration to the spermatozoon tail and middle piece region.

• Animal cells and systems
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• v.12 no.4
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• pp.297-304
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• 2008

Morphology and distribution of the minute tubercles projected on the skin surface of larvae with its development were observed in the Korean bitterling, Acheilognathus somjinensis. The minute tubercles appeared to be two distinct morphologies, hemispheric or scaly and vestigial structures. Just after hatching, the epidermis of the larvae consists of a thin single cell layer having smaller basophilic flat or round-flattened basal cells. As the larvae grow, the epidermis contains more small flat cells and large epidermal cells which are round and hemispheric, or scaleshaped, called minute tubercles. They are distributed over the anterior part and most part of yolk sac, posterior region of yolk sac and the body region. Vestigial epidermal cells, another minute tubercle, occur only in the caudal fin-fold region, which they are shrunken and flattened, causing the cell boundary to be unclear. They increase in number and height from just to 5 days after hatching, but they become reduced as the larvae develop gradually. The required time for those disappearance was different each by regional body: at day 20 after hatching in the anteriormost part of yolk sac, and day 11 after hatching in the posterior part of yolk sac and the body, and day 21 after hatching in two regions such most part of the yolk sac and the caudal finfold regions.

• Animal cells and systems
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• v.12 no.4
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• pp.193-201
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• 2008

The eventual goal of tumor immunotherapy is to develop a vaccine inducing a specific anti-tumor immunity. Cytokine gene therapy is an effective way at least in animal models, but limited efficacy and various side effects obstruct clinical applications. In this study, we developed a tumor vaccine expressing a membrane-bound form of IL-2(mbIL-2) and SDF-1 in B16F10 melanoma cells. The tumor clones expressing mbIL-2 showed reduced tumorigenicity, and additional expression of SDF-1 to mbIL-2 expressing tumor cells caused more severe reduction in tumorigenicity. However, expression of the SDF-1 alone did not affect on the tumorigenicity, probably because of limited production of SDF-1 in the SDF-1 transfected clones. When the mice once rejected mbIL-2/SDF-1 expressing tumor clone were re-challenged with wild type B16F10 tumor cells, all of the mice survived. This result suggests that mbIL-2/SDF-1 tumor clone is effective in inducing systemic anti-tumor immunity against wild type B16 melanoma. Furthermore, culture supernatant of tumor clones expressing SDF-1 induced lymphocyte migration in vitro. These results, all together, suggest that expression of mbIL-2 and SDF-1 in tumor cells enhances anti-tumor immune responses through different roles; the secreted SDF-1 may function as a chemoattractant to recruit immune cells to tumor vaccine injection site, and the mbIL-2 on tumor cells may provide costimulatory signal for CTL activation in physical contacts.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.18-23
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• 2007

Fe65, a neuron-specific adaptor protein, has two phosphotyrosine binding (PTB) domains. The second PTB (PTB2) domain interacts with intracellular domain fragment (AICD) of amyloid beta precursor protein (APP). Recent studies suggested that tile complex is composed of AICD and Fe65 transactivates genes that are responsible for neuronal cell death in Alzheimer's disease (AD). Therefore, a compound inhibiting the interaction between Fe65 and AICD can be a drug candidate to treat AD. However, it remains unclear how Fe65 recognizes AICD at a molecular level. Here, we report high-level production of the PTB2 domain of Fe65 in the baculovirus system. We found that the baculovirus system is an efficient method to obtain the Fe65 PTB2 domain, compared with the bacterial and mammalian expression systems. The purified recombinant protein was used for crystallization to determine its crystal structure helping to understand the molecular mechanism of Fe65-dependent signaling and to design its inhibitors.

• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.59-69
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• 2014

One of the major challenges in metabolic engineering for enhanced synthesis of value-added chemicals is to design and develop new strains that can be translated into well-controlled fermentation processes using bioreactors. The aim of this study was to assess the influence of various fed-batch strategies in the performance of metabolically engineered Pseudomonas putida strains, ${\Delta}gcd$ and ${\Delta}gcd-pgl$, for improving production of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) using glucose as the only carbon source. First we developed a fed-batch process that comprised an initial phase of biomass accumulation based on an exponential feeding carbon-limited strategy. For the mcl-PHA accumulation stage, three induction techniques were tested under nitrogen limitation. The substrate-pulse feeding was more efficient than the constant-feeding approach to promote the accumulation of the desirable product. Nonetheless, the most efficient approach for maximum PHA synthesis was the application of a dissolved-oxygen-stat feeding strategy (DO-stat), where P. putida ${\Delta}gcd$ mutant strain showed a final PHA content and specific PHA productivity of 67% and $0.83g{\cdot}l^{-1}{\cdot}h^{-1}$, respectively. To our knowledge, this mcl-PHA titer is the highest value that has been ever reported using glucose as the sole carbon and energy source. Our results also highlighted the effect of different fed-batch strategies upon the extent of realization of the intended metabolic modification of the mutant strains.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.357-364
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• 2017

In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, $4{\times}FLAG$, or $6{\times}HA$, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, using $Cac1-6{\times}HA$ and $Aca1-4{\times}FLAG$ tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using $aca1{\Delta}$::ACA1-GFP and $aca1{\Delta}$::ACA1-GFP $cac1{\Delta}$ strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.

• Journal of Microbiology and Biotechnology
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• v.27 no.7
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• pp.1345-1358
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• 2017

The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N-acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.