• IMMUNE NETWORK
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• v.11 no.3
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• pp.163-168
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• 2011

Background: Nanoparticles (NPs) prepared from biodegradable polymers, such as poly (D,L-lactic acid-co-glycolic acid) (PLGA), have been studied as vehicles for the delivery of antigens to phagocytes. This paper describes the preparation of antigen-loaded PLGA-NPs for efficient cross-priming. Methods: NPs containing a similar amount of ovalbumin (OVA) but different sizes were produced using a micromixer-based W/O/W solvent evaporation procedure, and the efficiency of the NPs to induce the cross-presentation of OVA peptides were examined in dendritic cells (DCs). Cellular uptake and biodistribution studies were performed using fluorescein isothiocyanate (FITC)-loaded NPs in mice. Results: The NPs in the range of $1.1{\sim}1.4{\mu}m$ in size were the most and almost equally efficient in inducing the cross-presentation of OVA peptides via $H-2K^b$ molecules. Cellular uptake and biodistribution studies showed that opsonization of the NPs with mouse IgG greatly increased the percentage of FITC-positive cells in the spleen and lymph nodes. The major cell type of FITC-positive cells in the spleen was macrophages, whereas that of lymph nodes was DCs. Conclusion: These results show that IgG-opsonized PLGA-NPs with a mean size of $1.1{\mu}m$ would be the choice of biodegradable carriers for the targeted-delivery of protein antigens for cross-priming in vivo.

• Development and Reproduction
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• v.11 no.1
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• pp.21-29
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• 2007

Previously, we found progesterone receptor membrane component 2 (pgrmc2) was highly expressed in germinal vesicle (GV) stage oocytes. The present study was conducted to characterize the expression of pgrmc2, as well as pgrmc1, in the mouse gonads and embryos according to their developmental stages. We found that these membrane components were expressed in ovaries, testes, and embryos at various developmental stages in addition to oocytes. Progesterone-3-O-carboxymethyl oxime-BSA-fluorescein isothiocyanate (P4-BSA-FITC) was applied to visualize the presence of the progesterone receptor on mouse oocyte membrane, and we confirmed that immobilized progesterone is localized at surface of the oocyte. This is, at our knowledge, the first report regarding the expression of membrane component of progesterone receptor in the mouse oocytes, embryos, and gonads. The function and signal transduction pathway of progesterone receptor membrane components in oocytes requires further studies.

• Korean Journal of Animal Reproduction
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• v.12 no.1
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• pp.37-41
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• 1988

These studies were carried out to examine the sex of preimplantation mouse embryo. For the investigation of sex-ration of mouse embryos, morula and blastocysts stage embryos treated with H-Y antiserum (10%, v/v) and FITC anti-mouse-IgG were divided into the positive and negative embryos. Positive and negative identified embryos were observed the viability according to the in vitro cultured and the sex ratio was also investigated by chromosomal analysis. The results obtained in these studies were summarized as follows: 1. Two hundred sixty-seven recovered embryos of morula or blastocyst stage were incubated in medium containing H-Y antiserum and FITC anti-mouse-IgG. Positively or negatively identified embryos were 139 and 128. This trend indicated the approximal sex ratio was 1:1. 2. Sex ratio was measured using the embryos treated with indirect immunofluorescence assay to examine the relationship between embryo developmental stage and sex ratio. Sex ratio of morula stage embryos was 45.2% positive and 54.8% negative, on the other hand, the ratio switched to 56.4% positive and 43.6% negative embryo in blastocyst stage. 3. Fourty-seven positive and 57 negative embryos were obtained out of 104 morula stage embryos treated with indirect immunofluorescence assay. Survived positive or negative embryos during in vitro culture were 42 and 49, respectively out of 47 and 57 embryos. 4. The numbers of negative and positive embryos were 171 and 92 out of 163 blastocyst embryos which were incubated in the medium containing H-Y antiserum and FITC anti-mouse-IgG. The result of karyotype test showed the successful rate of sexing embryo is positive and negative embryos was63.0% (58/92) and 62.0% (44/71). The final female to male ratio within 58 positive embryos was 22.7:77.6, and the ratio of the 44 negative embryos was 77.3:22.7.

• Applied Chemistry for Engineering
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• v.20 no.6
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• pp.632-637
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• 2009

Poly(${\varepsilon}-caprolacton$)/ethyl cellulose (PCL/EC) microcapsules containing pluronic F127 were prepared by a spray drying method. The aqueous phase, 20% of pluronic F127 was dissolved in distilled water, and the organic phase, 5% of PCL and EC were dissolved in dichloromethane. The microcapsules were obtained by spray drying the water-in-oil (W/O) emulsion. According to the data of scanning electron microscopy and particle analyzer, tens of micro size microcapsules were observed. On a differential scanning calorimeter, the phase transition temperatures of microcapsules were observed and they were found around those of pluronic F127 and poly(${\varepsilon}-caprolacton$), which were the main components of the microcapsules. At the range of $30{\sim}45^{\circ}C$, temperature-dependent release properties were investigated using fluorescein isothicyanate-dextran (FITC-dextran) and blue dextran as a model drug. When the temperature was increased, the degree of release of microcapsule was also increased. FITC-dextran, the relative low molecular weight, was more released than blue-dextran.

• KSBB Journal
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• v.22 no.4
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• pp.260-264
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• 2007

Peptides are frequently studied as candidates for new drug development. Recently, synthesized peptide library is screened for a certain functionality on a microarray biochip format. In this study, in order to replace the conventional cellulose membrane with glass for a microarray chip substrate for peptide library screening, we modified the glass surface from amines to thiols and covalently immobilized the peptides. Using trypsin-FITC (fluorescein isothiocyanate) conjugate that could specifically bind to a trypsin binding domain consisting of a 7-amino acid peptide, we checked the degree of surface modification. Because of the relatively lower hydrophilicity and reduced surface roughness, the conjugation reaction to the glass required a longer reaction time and a higher temperature. It took approximately 12 hr for the reaction to be completed. From the fluorescence signal intensity, we could differentiate between the target and the control peptides. This difference was confirmed by a separate experiment using QCM. Furthermore, a smaller volume and higher concentration of a spot showed a higher fluorescence intensity. These data would provide the basic conditions for the development of microarray peptide biochips.

• Microbiology and Biotechnology Letters
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• v.30 no.3
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• pp.235-240
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• 2002

Streptomycetes is a group of Gram-positive soil bacteria that growas a branching vegetative mycelium leading to the formation of spores, and display a physiological differenti-ation related to the synthesis of many secondary metabolites including antibiotics. Their complex life cycle and multicellular differentiation require various levels of regulation and types of signal transduction systems including eukaryotic-type serine/threonine protein kinases and prokaryotic-type histidine/aspartic acid protein kinases. Akt kinase that was found in cells is a sorine/threonine kinase controlling signal pathway for multi-tude of important cellular events. The activation or inactivation of Akt kinase in the cell is one of the critical regulatory points to deliver cell proliferation, differentiation, survival or apoptosis signal. To find the regula-tory protein homologous to Akt in Streptomyces, the fluorescien-labeled synthetic peptide (FITC-TRRSR-TESIT) was designed from the consensus sequence of target proteins for Akt kinase. From the difference of the mobility between the nonphosphorylated and phosphorylated synthetic peptides on Agarose gel electro-phoresis, the Akt-phosphorylating activity was monitored. The cell-free extract prepared from Streptomyces griseus IFO 13350 and the Akt homologous protein was purified by ammonium sulfate fractionation and many steps of column chromatographies such as, DEAE-Sepharose, Mono Q, Resource Phenyl-Soporose and Gel permeation column chromatographies. As a result, the protein phosphorylating the fluorescien-labeled Akt substrate was identified and it's molecular weight was estimated as 39 kDa on SDS-PAGE.

• Applied Microscopy
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• v.33 no.2
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• pp.105-116
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• 2003

In Vitro fertilization of U. unicinctus eggs observed by immunofluorescence and electron microscopes revealed an overview of the meiotic pattern of the tide animals. The eggs have been fertilized early at germinal vesicle stage, followed by germinal vesicle break down (GVBD), but pre-mitotic aster like structure could not be resolved by the methods employed in this work. The meiotic features, such as rotation-shift movement of spindle fibers, behavior of spermatozoonmonaster in the egg cytoplasm and active spindle fiber of the 1st polar body, have been observed. The antitubulin-FITC fluorescence show the 2nd meiotic apparatus appeared firstly parallel to the tangential line of the oolemma, proceeding the meiosis, its bipolarity is rotated and shifted towards the oolemma. The polar bodysite of the oolemma was not amorphous, but active in a sense of anti-tubulin-FITC reactions during the extrusions of the polar bodies. The immunofluorescence reactions of the spermatozoon centriole appeared at a later stage of the 2nd meiosis. During the time periods, the fertilized spermatozoon resided in the egg cytoplasm. Activating the centrioles, spermatozoon approaches towards the chromosomal materials of the 2nd oocyte. This suggests that spermatozoon centrioles initiate and play a roll to fuse male and female pronuclei.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.2885-2889
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• 2013

Goniothalamin is an active compound extracted from Goniothalamus griffithii, a local plant found in northern Thailand. Goniothalamin inhibits cancer cell growth but is also toxic to normal cells. The aims of this study were to identify the cytotoxic effect of goniothalamin and the mechanism of cell death in human HL-60 and U937 cells. Cytotoxicity was determined by MTT assay and cell cycle profiles were demonstrated by staining with propidium iodide (PI) and flow cytometry. Apoptosis was confirmed by staining with annexin V-FITC/propidium iodide (PI) and flow cytometry. Reduction of mitochondrial transmembrane potential was determined by staining with dihexyloxacarbocyanine iodide and flow cytometry and expression of Smac, caspase-8 and -9 was demonstrated by Western blotting. Goniothalamin inhibited growth of HL-60 and U937 cell lines. An increase of SubG1 phase was found in their cell cycle profiles, indicating apoptosis as the mode of cell death. Apoptosis was confirmed by the flip-flop of phosphatidylserine using annexin V-FITC/PI assay in HL60 and U937 cells in a dose response manner. Furthermore, reduction of mitochondrial transmembrane potential was found in both cell types while expression of caspase-8, -9 and Smac/Diablo was increased in HL-60 cells. Taken together, our results indicate that goniothalamin-treated human leukemic cells undergo apoptosis via intrinsic and extrinsic pathways.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.318-321
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• 2019

Recent studies showed that tight junctions (TJs) integrity and assembly are required for blastocyst development in mouse and pig models. However, the biological functions of TJs associated with embryo implantation and maintenance of pregnancy were not investigated yet. To examine whether disrupted TJs affect further embryo development, we employed RNAi approach and inhibitor treatment. The embryos were injected with Cxadr (Coxsackievirus and adenovirus receptor) siRNA for knock down (KD) and treated with Adam10 (A Disintegrin and Metalloproteinase specific inhibitor 10; GI254023X; SI). We compared blastocyst development and paracellular sealing assay using FITC dextran uptake between control and KD or SI embryos. Finally, we transferred control and Cxadr KD or Adam 10 SI treated blastocyst to uteri of recipients. Cxadr KD and Adam 10 SI showed lower blastocyst development and more permeable to FITC-dextran. Moreover, we observed that half of KD and inhibited embryos failed to maintain pregnancies after the second trimester. Our findings suggested that TJs integrity is required for the maintenance of pregnancy and can be used as a selective marker for the successful application of assisted reproduction technologies.

• YAKHAK HOEJI
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• v.55 no.1
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• pp.49-55
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• 2011

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is one of the promising anti-cancer agent because of its ability to selectively induce apoptosis in tumor cell lines but not in normal cells. However, TRAIL resistance has been reported in some cancer cells including hepatocarcinoma cells. Therefore, studies of agents that sensitize TRAIL-resistant cancer cells could be a effective therapeutic approach in cancer management. In our study, we examined the effect of combination of TRAIL with apigenin in human hepatocellular carcinoma cells. As a result, the combined use of TRAIL and apigenin significantly enhanced the cytotoxicity in PLC-PRF5 cells. Flow cytometry analysis after annexin V-FITC/PI dual staining showed that this increase of cell cytotoxicity was related to enhanced apoptosis in combined treatment of TRAIL with apigenin. Furthermore, synergistic induction of apoptosis was also confirmed by observation of morphological changes and annexin V-FITC/PI fluorescence. Our findings suggests that apigenin has the potential to improve the efficiency of TRAIL-based therapies in human hepatocellular carcinoma cells. Further study is needed to reveal the molecular mechanisms of this combined therapy.