• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.918-922
• /
• 2001

In a previous study, we have isolated a number of lactobacilli from Korean women, and one of them (KLB46) was identified as Lactobacillus crispatus by 16S rRNA gene sequencing. For the ecological treatment of bacterial vaginosis (BV) cell suspension of L. crispatus KLB46 was instillated into BV patients. L. crispatus KLB46 was found to persist for several days in cell suspension with no nutrients. In this study, in order to assess the influence of starvation on physiological activity, we compared the viability and culturability of KLB46 following suspension in various buffer solutions. A pair of in situ fluorescent dye was used to assess viability (i.e. membrane integrity) and the culturability was examined by plate count assay. A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit $(BacLight^{TM})$ was applied to estimate both viable and total counts of bacteria in cell suspension. $BacLight^{TM}$ is composed of two nucleic acid-binding stains ($SYTO\;9^{TM}$ and propidium iodide). $SYTO\;9^{TM}$ penetrates all bacterial membranes and stains the cells green while propidium iodide only penetrates cells with damaged membranes, therefore the combination of the two stains produces red fluorescing cells. Optimal staining conditions for $BacLight^{TM}$ were found to be with 0.0835M $SYTO\;9^{TM}$ and 0.05M propidium iodide for 15 min incubation at room temperature in dark. When cells were microscopically examined during 140 hours of starvation, the culturability decreased markedly while the viability remained relatively constant, which suggests that large fraction of KLB46 cells became viable but non-culturable (VBNC) upon starvation.

• The Journal of Korean Medicine
• /
• v.39 no.1
• /
• pp.35-43
• /
• 2018

Objectives: In the brain, glutamate is the most important excitable neurotransmitter in physiological and pathological conditions. However, the high level of glutamate induces neuronal cell death due to exitotoxicity and oxidative stress. The present study investigated to evaluate a possible neuroprotective effect of furosin isolated from Euphorbia helioscopia against glutamate-induced HT22 cell death. Methods: Furosin was isolated from methanol extract of Euphorbia helioscopia and examined whether it protects glutamate-induced neuronal cell death. The cell viability was determined using Ez-Cytox assay. Anti-oxidative effect of furosin was determined by DPPH scavenging activities, and the levels of intracellular reactive oxygen species (ROS) were determined by the fluorescent intensity after staining the cells with $H_2DCFDA$. To evaluate apoptotic cell death, we performed nuclear staining and image-based cytometeric analysis. Results: The cell viability was significantly increased by treatement with furosin compared with the treatment with glutamate. Furosin showed a strong DPPH radical scavenging activity ($EC50=1.83{\mu}M$) and prevented the accumulation of intra cellular ROS. Finally, the presence of 50 and $100{\mu}M$ furosin significantly the percentage of apoptotic cells compared with glutamate treatment. Conclusion: The present study found that furosin is a potent neuroprotectant against glutamate-induced oxidative stress through inhibition of apoptotic cell death induced by glutamate. Therefore, the present study suggests that furosin as a bioactive compound of E. helioscopia can be a useful source to develop a drug for the treatment of neurodegenerative diseases and acute brain injuries.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.12
• /
• pp.6221-6225
• /
• 2012

Background: To determine the frequency of HER-2 overexpression in colorectal cancer (CRC) patients, and to explore the relationship between clinicopathological prognostic factors and their effects on survival, based on immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) analysis. Materials and Methods: The study included 80 patients with a histologically proven diagnosis of CRC that received adjuvant FOLFOX-4 chemotherapy at our department between March 2006 and September 2010. Patient data were analyzed retrospectively. Results: The median follow-up period and age of the patients were 24 months and 59 years, respectively. In immunohistochemical staining, 3+ staining was found in 2 patients (2.5%) while 2+ was in 13 (16%). FISH for HER-2 was performed for all of these 15 patients; samples which were 3+ showed positivity but the ones with 2+ were negative. There was no significant correlation between HER-2 expression and age, gender, tumor localization, histological subtype, grade, lymphovascular and perineural invasion, or pTN stage (P>0.05), even when the patients with HER-2 overexpression were analyzed separately. There was also no significant relationship between progression-free survival (PFS) and overall survival (OS), and HER-2 expression, gender, tumor localization, obstruction-perforation, bleeding, histological type, grade, lymphovascular and perineural invasion, or pT staging (P>0.05); however, there was a significant relationship between lymph node involvement, and PFS and OS (P<0.05). Conclusions: Evaluation of HER-2 overexpression in a more comprehensive, multi-center, prospective trial with standardized methods will be an appropriate approach.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.12
• /
• pp.6211-6216
• /
• 2012

Objective: To investigate the effects of gambogic acid (GA) on the growth of human malignant glioma cells. Methods: U251MG and U87MG human glioma cell lines were treated with GA and growth and proliferation were investigated by MTT and colony formation assays. Cell apoptosis was analyzed by annexin V FITC/PI flow cytometry, mitochondrial membrane potential assays and DAPI nuclear staining. Monodansylcadaverine (MDC) staining and GFP-LC3 localisation were used to detect autophagy. Western blotting was used to investigate the molecular changes that occurred in the course of GA treatment. Results: GA treatment significantly suppressed cell proliferation and colony formation, induced apoptosis in U251 and U87MG glioblastoma cells in a time- and dose-dependent manner. GA treatment also lead to the accumulation of monodansylcadaverine (MDC) in autophagic vacuoles, upregulated expressions of Atg5, Beclin 1 and LC3-II, and the increase of punctate fluorescent signals in glioblastoma cells pre-transfected with GFP-tagged LC3 plasmid. After the combination treatment of autophagy inhitors and GA, GA mediated growth inhibition and apoptotic cell death was further potentiated. Conclusion: Our results suggested that autophagic responses play roles as a self-protective mechanism in GA-treated glioblastoma cells, and autophagy inhibition could be a novel adjunctive strategy for enhancing chemotherapeutic effect of GA as an anti-malignant glioma agent.

• Biomedical Science Letters
• /
• v.13 no.4
• /
• pp.273-278
• /
• 2007

Stem cells have been the subject of increasing scientific interest because of their utility in numerous biomedical applications. Stem cells are capable of renewing themselves; that is, they can be continuously cultured in an undifferentiated state, giving rise to more specialized cells of the human body. Therefore, stem cells are an important new tools for developing unique, in vitro model systems to test drugs and chemicals and a potential to predict or anticipate toxicity in humans. In the present study, in vitro cultured F3 immortalized human neural stem cell line and in vivo adult Sprague Dawley rats was used to evaluate the cytotoxicity of anticancer drug paclitaxel. In vitro apoptotic activity of paclitaxel was evaluated in F3 cell line by a MTT assay and DAPI test. The cell death was induced with the treatment of 20 nM paclitaxel and chromatin degradation was detected by DAPI staining, which was analyzed by fluorescent microscope. In vivo studies, we also observed nestin immunoreactivity on subventricular zone, which is stem cell rich region in the adult brain of the SD rat. Immunofluorescent staining result shows that pixel intensities of nestin were decreased in a dose dependent manner. These results suggest that paclitaxel is able to induce cytotoxic activity both in F3 neural stem cell line and neural stem cell in SD rat brain.

• ALGAE
• /
• v.32 no.1
• /
• pp.15-28
• /
• 2017

Morphological, molecular and chromosomal studies were carried out on Tauya basicrassa, an endemic kelp species distributed on the northern continental coast of the Sea of Okhotsk in Russia. The sporophytes of T. basicrassa grow up to 3-6 m long, 1.8-2.2 m wide, and 6.5-7 kg wet weight. The thallus has a blade with very thick narrow basal portion and thinner and much broader upper portion, which usually splits into 3 bullated lobes. A dwarf laminariacean alga, which did not show any morphological similarity to the other species of the order Laminariales, was found from the same locality. The blade of this alga is thin and soft, reached 26-34 cm long and 6-6.5 cm wide and had 4 longitudinal rows of bullations that covered the entire blade. Molecular analysis showed that the dwarf alga has 100% sequence identity in plastid-encoded RuBisCo spacer, mitochondrial cytochrome c oxidase subunit 1 and nuclear-encoded rDNA genes with normal sporophytes of T. basicrassa, indicating that they are different life forms of the same species. Fluorescent DAPI staining showed that the nucleus in the normal sporophyte was 50-65% larger than those of the dwarf ones. Chromosome count using acetocarmine staining showed n = ca. 20 for the normal sporophytes of T. basicrassa and n = ca. 10 for the dwarf one. These results suggest that the dwarf thallus is a haploid parthenosporophyte of T. basicrassa, which developed in nature. This is the first evidence of parthenosporophytes of the laminariacean algae occurring naturally in the field.

• ALGAE
• /
• v.34 no.2
• /
• pp.71-90
• /
• 2019

A new encrusting red alga was found growing abundantly on glass debris items that drifted ashore along the coasts of Oregon and Washington. These included discarded fluorescent tubes, incandescent light bulbs, capped liquor bottles, and ball-shaped fishing-net floats. Field collections and unialgal cultures of the alga revealed that it consisted of two morphological phases: a young loosely aggregated turf and a mature consolidated mucilaginous crust. The turf phase consisted of a basal layer of globose cells that produced erect, rarely branched, uniseriate to multiseriate filaments up to $500{\mu}m$ long with closely spaced cells lacking pit-plugs. These filaments expanded in size from their bases to their tips and released single cells as spores. At maturity, a second phase of growth occurred that produced a consolidated crust, up to $370{\mu}m$ thick. It consisted of a basal layer of small, tightly appressed ellipsoidal-to-elongate cells that generated a mucilaginous perithallial matrix containing a second type of filament with irregularly spaced cells often undergoing binary division. At the matrix surface, the original filaments continued to grow and release spores but often also eroded. Individual cells, examined using confocal microscopy and SYBR Green staining, were found to contain a central nucleus, a single highly lobed peripheral chloroplast without a pyrenoid, and numerous chloroplast nucleoids. Morphological data from field and culture isolates and molecular data (rbcL, psbA, and SSU) show that this alga is a new genus and species which we name Viator vitreocola, "a traveller on glass."

• Journal of Animal Reproduction and Biotechnology
• /
• v.35 no.4
• /
• pp.347-356
• /
• 2020

Gangliosides are glycolipids in which oligosaccharide is combined with sialic acids. Our previous studies have suggested an interplay between ganglioside GD1a/GT1b and meiotic maturation capacity in porcine oocyte maturation. Furthermore, ganglioside GD1a and GT1b are known for its antioxidant activity, but it is still unclear whether possible antioxidant role of GD1a and GT1b is involved in porcine embryos development competence during in vitro culture (IVC). Here, the effects of ganglioside GD1a and GT1b on the embryonic developmental competence during in vitro culture of porcine were investigated. The effects of ganglioside GD1a and GT1b on the expression of ST3GAL2 were confirmed during embryos development (2-cell, 4-cell, 8-cell and blastocyst) using immunofluorescent staining (IF). As a result, the fluorescent expression of ST3GAl2 was higher in embryos at 4-8 cells stage than blastocysts. Blastocyst development rate significantly increased in only 0.1 μM GD1a and GT1b treated groups compared with control group. To investigate the cellular apoptosis, we analyzed TUNEL assay. In case of only 0.1 μM GD1a and GT1b treated groups, the total number of cells in blastocyst compared with control group, but there was no significant difference in the rate of apoptotic cells. We identified the intracellular ROS levels using DCF-DA staining. According to the result, ROS production significantly decreased in blastocysts derived from the 0.1 μM GD1a and GT1b treated groups. These results suggest that ganglioside GD1a and GT1b improve the developmental competence of porcine embryos via reduction of intracellular ROS during preimplantation stage.

• Clinical and Experimental Pediatrics
• /
• v.51 no.10
• /
• pp.1112-1117
• /
• 2008

Purpose : We intended to observe cell death and apoptotic changes in neurons in organotypic hippocampal slice cultures following oxygen-glucose deprivation (OGD), using propidium iodide (PI) uptake, Fluoro-Jade (FJ) staining, TUNEL staining and immunofluorescent staining for caspase-3. Methods : The hippocampus of 7-day-old rats was cut into $350{\mu}m$ slices. The slices were cultured for 10 d (date in vitro, DIV 10) and and exposed to OGD for 60 min at DIV 10. They were then incubated for reperfusion under normoxic conditions for an additional 48 h. Fluorescence of PI uptake was observed at predetermined intervals, and the cell death percentage was recorded. At 24 h following OGD, the slices were Cryo-cut into $15{\mu}m$ thicknesses, and Fluoro-Jade staining, TUNEL staining, and immunofluorescence staining for caspase-3 were performed. Results : 1) PI uptake was restricted to the pyramidal cell layer and DG in the slices after OGD. The fluorescent intensities of PI increased from 6 to 48 h during the reperfusion stage. The cell death percentage significantly increased time-dependently in CA1 and DG following OGD (P<0.05). 2) At 24 h after OGD, many FJ positive cells were detected in CA1 and DG. Some neurons had distinct nuclei and processes while others had fragmented nuclei and disrupted processes in CA1. TUNEL and immunofluorescent staining for caspase-3 showed increased expression of TUNEL labeling and caspase-3 in CA1 and DG at 24 h after OGD. Conclusion : The numerous dead cells in the slice cultures after OGD tended to display apoptotic changes mediated by the activation of caspase-3.

• Proceedings of the Korean Society of Precision Engineering Conference
• /
• 2004.10a
• /
• pp.1215-1220
• /
• 2004

Cell adhesion to any material surface is considered to be fundamental and important phenomenon in the fields of tissue engineering. Cell adhesion molecules, mechanism, and attachment force have been studied and described a lot. However, the effects of mechanical stimuli on the adhesive forces still have been left much to be investigated. In this study, to investigate the changes in cell adhesive force due to resting time period during the intermittent hydrostatic pressurizing (IHP), cells were cultured under the IHP with various resting times. Then the cell adhesive forces were measured quantitatively utilizing a cell detachment test system and immunofluorescent staining was performed using fluorescent microscopy. In the results, immediately after mechanical stimuli (150 minutes after seeding) and one hour later (210 minutes after seeding), the average adhesive force of experimental group 5 (resting time: 15min) compared with that of control group at same culture time was increased significantly (p<0.05). The results indicated that IHP can contribute in improving cell adhesive force and some of time intervals were required for the expression of cell response.