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ABSTRACT. Gap junctional intercellular communications (GJIC) contributes to neural function in development and differentiation of CNS. In this study, we have investigated the expression of GJIC during the differentiation of neuronal stem cells and 12-Ø-tetradecanoylphorbol-13-acetate (TPA)-induced neuronal stem cell-derived cells from rat brain. During neuronal stem cell differentiation, expressions of Cx43 and 32 were increased for the duration of 72 hr, however the effect were decreased on the 7d. In the neuronal stem cell-derived cells, pretreatments with p38 MAP kinase inhibitor, SB203580, and MEK inhibitor, PD98059, could protect GJIC against TPA-induced inhibition of GJIC. Our data suggest that GJIC plays an important role during neuronal stem cell differentiation, and ERK1/2 and p38 MAP kinase signaling pathway may be closely related functionally to regulate gap junction in rat neuronal stem cell-derived cells. Neuronal stem cells are able to differentiate into astrocytes, neurons, and oligodendrocytes [3,10,23], when given the appropriate signals [2,7]. During the stage where the brain develops, gap junctional intercellular communication (GJIC) allows the diffusional exchange of ions and metabolites for communication between neighboring cells [8,14,20]. Connexin 32 and 43 were expressed in the neurons of adult rat or mouse [15,17], and Cx43 was the most abundant gap junction protein in the CNS [4,5]. However, no study has yet been performed to the role of gap junctions in rat neuronal stem cell differentiation. In the present study, we have examined protein expression of Cx43 and Cx32 during the neuronal stem cell differentiation and induction of Cx43 and Cx32. This can be elicited in neuronal stem cell-derived cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and this induction showed that it can be prevented by the p38 MAP-Kinase inhibitor SB203580 and the MEK inhibitor PD98059. MATERIALS AND METHODSCell culture: Neuronal stem cells were prepared by culturing cells from cerebrum of embryonic day 17 (E17) Sprague-Dawley rat (Bio Genomics, Korea) fetus. Briefly, fresh cerebrums were dissociated in the presence of trypsin and DNase I and planted on a Petri dish (Nunc, IL). Cells were seeded at a density of 1~5 × 10 6 cells/ml in a mixture (1:1) of Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12; Gibco) replenished with 2% B27 (Gibco), gentamycin (50 µg/ml), anti-PPLO (pleuro-pneumonia-like organism) reagent (100 µg/ml), recombinant human basic fibroblast growth factor (bFGF 20 ng/ml, Sigma), and epidermal growth factor (10 ng/ml).Scrape loading/ dye transfer (SL/DT) assay: Cells were treated with TPA for 1 hr. The GJIC assay was conducted at non-cytotoxic dose levels of the samples, as determined by the MTT assay. Following incubation, the cells were washed twice with 2 ml of PBS, Lucifer yellow was added to the washed cells and three scrapes were made with a surgical steel-bladed scalpel at low light intensities. These three scrapes were performed to ensure that the scrape travers...

A few starters have been developed and used for doenjang fermentation but often without safety evaluation. Filamentous fungi were isolated from industrial doenjang koji, and their potential for mycotoxin production was evaluated. Two fungi were isolated; one was more dominantly present (90%). Both greenish (SNU-G) and whitish (SNU-W) fungi showed 97% and 95% internal transcribed spacer sequence identities to Aspergillus oryzae/flavus, respectively. However, the SmaI digestion pattern of their genomic DNA suggested that both belong to A. oryzae. Moreover, both fungi had morphological characteristics similar to that of A. oryzae. SNU-G and SNU-W did not form sclerotia, which is a typical characteristic of A. oryzae. Therefore, both fungi were identified to be A. oryzae. In aflatoxin gene cluster analysis, both fungi had norB-cypA genes similar to that of A. oryzae. Consistent with this, aflatoxins were not detected in SNU-G and SNU-W using ammonia vapor, TLC, and HPLC analyses. Both fungi seemed to have a whole cyclopiazonic acid (CPA) gene cluster based on PCR of the maoA, dmaT, and pks-nrps genes, which are key genes for CPA biosynthesis. However, CPA was not detected in TLC and HPLC analyses. Therefore, both fungi seem to be safe to use as doenjang koji starters and may be suitable fungal candidates for further development of starters for traditional doenjang fermentation.

초록: 망막 조직공학을 위한 생체 재료는 기계적 안전성, 생체적합성, 낮은 분해속도 등을 포함하여 in vivo에서 잠 재적인 유용성을 위한 몇 가지 중요한 특징이 입증되어야 한다. 실크 필름 생체재료는 이러한 기능적인 요구에 맞게 디자인되었다. 0, 10, 20, 40, 및 80 wt%의 실크가 함유된 천연/합성물질과 하이브리드화된 silk/PLGA 필름을 용매 증발법으로 제조하였다. 1, 2, 및 3일 후에 부착된 세포 수를 확인하기 위해 MTT 분석을 하였고 SEM을 통해 필름 에 부착된 세포 모폴로지를 확인하였다. 또한, mRNA 발현정도를 알아보기 위해 retinal pigment epithelium(RPE) 세 포의 프라이머인 RPE65를 사용하여 RT-PCR을 실시하였고 RPE 세포의 특정 단백질인 cytokeratin의 발현을 확 인하고 세포의 증식을 비교하기 위해 면역화학염색을 실시하였다. 본 실험을 통해 실크/PLGA 필름에서 20∼40 wt% 실크를 함유한 경우에 RPE 세포의 부착과 증식에 가장 좋은 영향을 미치는 것을 확인하였다.Abstract: Biomaterials for retinal tissue engineering must demonstrate several critical features for potential utility, including mechanical integrity, biocompatibility, and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. We prepared natural/synthetic hybrid silk/PLGA films using 0, 10, 20, 40, and 80 wt% of silk by a solvent evaporation method. MTT assay was used to confirm the number of cells attached on film at 1, 2, and 3 days, respectively. The morphology of cellular adhesion on films was also confirmed by scanning electron microscope (SEM). RT-PCR was conducted to confirm mRNA expression of retinal pigment epithelium(RPE) using RPE65 as a RPEs marker and the expression of cytokeratin were determined by immunofluorescence staining. We confirmed that the silk/PLGA film of 20∼40 wt% silk was superior for the adhesion and proliferation of RPEs.

The retinal pigment epithelium (RPE) closely interacts with photoreceptors in the maintenance of visual function. The native RPEs exist as a monolayer structure and have a mottled brown color due to the presence of melanin and other pigments including lipofuscin granules, which accumulate with age. In age-related macular degeneration (AMD), RPE's dysfunction and changes in Bruch's membrane occur. Thus, small intestine submucosa/poly(lactic-co-glycolic acid) (SIS/PLGA) film is a biomimetic transplant consisting of a layer of healthy RPE cells cultured on a support membrane. The goals of this study were to evaluate the effects of attachment and proliferation of RPEs on SIS/PLGA films. Porcine SIS is an acellular tissue and widely used as a biomaterial without immunorejection responses, whereas PLGA is a biodegradable synthetic polymer with acceptable mechanical strength and well-controlled degradation rate. We fabricated SIS/PLGA films using 20 wt% of SIS. We measured MTT to confirm cellular adhesion of cell number attached on film at 1, 3, 5, and 7 days. Morphology of cellular adhesion on films was confirmed by scanning electron microscopy at 1, 3, and 7 days. Further, reverse transcription polymerase chain reaction (RT-PCR) was conducted to confirm messenger RNA expression of RPE65 as RPE's marker and expression of cytokeratin, and RPE65 were determined by AEC immunocytochemical staining. These results suggest that SIS provides suitable surface to RPEs.

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