• Clinical and Experimental Reproductive Medicine
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• v.32 no.3
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• pp.207-216
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• 2005

Objective: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Müllerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. Materials and Methods: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in ${\alpha}$-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. Results: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. Conclusion: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

• Parasites, Hosts and Diseases
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• v.52 no.5
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• pp.493-499
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• 2014

The determination of the accurate immune status of pregnant women is crucial in order to prevent congenital toxoplasmosis. Equivocal results with conventional serological techniques are not uncommon when IgG titers are close to the cut-off value of the test, so that a confirmatory technique is needed. For this purpose, we developed a homemade immunoblot (IB) using soluble extract of Toxoplasma gondii tachyzoites and assessed it by testing 154 positive, 100 negative, and 123 equivocal sera obtained from pregnant women. In order to select the more valuable bands in terms of sensitivity and specificity, we used the Youden Index (YI). The highest YIs were those given by the 32, 36, 98, 21, and 33 bands. The simultaneous presence on the same blot of at least 3 bands showed a much higher YI (0.964) and was adapted as the positivity criterion. The analysis of results showed that our homemade IB correlated well with the commercial LDBIO Toxo II $IgG^{(R)}$ kit recently recommended as a confirmatory test (96.7% of concordance).

• Journal of the korean academy of Pediatric Dentistry
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• v.26 no.3
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• pp.466-472
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• 1999

The insoluble glucan is the major substance of dental plaque. In order to isolate the bacteria inhibiting the formation of insoluble glucan in disposable cuvette, saliva was got from about 10 thousand children. The isolated bacteria were tested by API 20S kit and API 50 CHL kit. These bactreia were identified as Streptococcus oralis, Streptococcus mitis, Streptococcus mitior, Streptococcus sanguis, Enterococcus durans, Lactococcus lactis, Lactobacillus acidophilus. When Streptococcus mutans was cultured with Streptococcus oralis, Streptococcus mitis, Streptococcus mitior, Streptococcus sanguis, Enterococcus durans, Lactococcus lactis, or Lactobacillus acidophilus in disposable cuvette, the optical density at 550 nm was 0.823, 0.912, 0.894, 0.878, 0.753, 0.845, 1.021 respectively, while being 1.503 in the disposable cuvette culturing Streptococcus mutans only.

• Parasites, Hosts and Diseases
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• v.52 no.3
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• pp.263-271
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• 2014

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. $QIAamp^{(R)}$ DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume ($50-100{\mu}l$) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ${\approx}2$ oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

• Journal of Genetic Medicine
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• v.12 no.1
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• pp.31-37
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• 2015

Purpose: We investigated the neurogenic potentials of amniotic fluid-derived stem cells (AFSCs) according to the expression levels of stem cell markers and ingredients in the neural induction media. Materials and Methods: Four samples of AFSCs with different levels of Oct-4 and c-kit expression were differentiated neurally, using three kinds of induction media containing retinoic acid (RA) and/or a mixture of 3-isobutyl-1-methylxanthine/indomethacin/insulin (neuromix), and examined by immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR) for their expression of neurospecific markers. Results: The cells in neuromix-containing media displayed small nuclei and long processes that were characteristic of neural cells. RT-PCR analysis revealed that the number of neural markers showing upregulation was greater in cells cultured in the neuromix-containing media than in those cultured in RA-only medium. Neurospecific gene expression was also higher in Oct-4 and c-kit double-positive cells than in c-kit-low or -negative cells. Conclusion: The stem cell marker c-kit (rather than Oct-4) and the ingredient neuromix (rather than RA) exert greater effects on neurogenesis of AFSCs.

• IMMUNE NETWORK
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• v.5 no.2
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• pp.124-129
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• 2005

Background: CM1 (centrocyte/-blast marker 1) is originally defined as a germinal center B cell marker. It is known that CM1 plays a critical role on B cell development in germinal center. In addition, we have found that CM1 is expressed on lymphoma cell lines, such as Raji, Ramos and IM-9. This means that CM1 might be served as a tumor marker as well. In the present study, we examined the expression of CM1 on the surface of the other tumors and the possibility of the development of tumor screening ELISA kit by using CM1. Methods: First, we have examined the expression of CM1 on stomach cancer and hepatoma, which are predominantly (discovered) occurred in Korean, by flow cytometry analysis. After purifying of CM1 antigen from Raji and Ramos, the optimal ELISA condition was determined. And then we compared the level of CM1 between normal individuals and cancer patients by ELISA. To decrease the non-specific binding of anti-CM1 mAb with serum components except CM1 and to enhance the diagnostic accuracy, albumin depletion spin column was used. Results: CM1 was highly expressed on stomach cancer and hepatoma cell lines. In addition, we have also confirmed the increased CM1 expression on cancer patients. The difference of CM1 expression between normal individuals and cancer patients were more clearly observed, after deletion of serum albumin by using albumin depletion spin column. Conclusion: Based on the results from this study, CM1 might be a useful molecule for the early diagnosis of cancer. In addition, further studies for the increase of ELISA sensitivity and appropriate albumin depletion methods should be needed.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2753-2758
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• 2014

Background: GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. Materials and Methods: GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. Results: Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. Conclusions: These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1851-1857
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• 2014

Objective: The main purpose of this work was to investigate the effect of berberine hydrochloride (BH) on the proliferation, apoptosis, migration, and invasion of CNE-1 nasopharyngeal carcinoma cells. Our results shed light on the functional components of traditional Chinese herbs for potential use in modern medicine. Methods: The CNE-1 cell line was treated with different concentrations of BH and effects on cell viability and proliferation were evaluated using the Cell Counting Kit-8 (CCK-8) assay. Anti-migratory and anti-invasive actions of BH were investigated using wound healing assays and the Millicell Hanging cell culture insert system, respectively. Expression of the epithelial-mesenchymal transition (EMT)-related gene twist (Twist) was analyzed by real-time PCR and Western blotting. Apoptosis was estimated with an annexin-V fluorescein (FITC) apoptosis detection kit, as well as with reference to levels of activated caspase-3 of CNE-1 cells before and after treatment with BH utilizing fluorescence spectroscopy. Results: BH was capable of reducing proliferation and viability of CNE-1 cells in a dose- and time-dependent manner, also demonstrating anti-migratory and anti-invasive capacities which correlated with reduction in expression of Twist. Finally, BH was able to induce significant amounts of apoptosis in CNE-1 cells, as demonstrated by an increase in the activity of caspase-3 and in annexin-V staining following treatment. Conclusion: BH extracted from rhizoma coptidis demonstrated an ability to block proliferation, induce apoptosis, and impair the migration and invasion of the CNE-1 cell line Considering these properties, our results suggest that BH could be an important compound for consideration in the treatment of nasopharyngeal carcinoma.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3179-3183
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• 2016

The aim of this study was to assess the rate of uptake of a customised annual Colorectal Cancer Awareness, Screening and Treatment Project (CCASTP) using faecal immunohistochemical test (FIT) kits in low income communities in Malaysia. The immediate objectives were (1) to evaluate the level of adherence of CRC screening among low-income groups, (2) to assess the knowledge and awareness of the screened population and (3) to assess the accuracy of FIT kits. A total of 1,581 FIT kits were distributed between years 2010 to 2015 to healthy asymptomatic participants of the annual CCASTP organized by Empowered - the Cancer Advocacy Society of Malaysia. Data for socio-demographic characteristics, critical health and lifestyle information of the registered subjects were collected. Findings for use of the FIT kits were collected when they were returned for stool analyses. Those testingd positive were invited to undergo a colonoscopy examination. A total of 1,436 (90.8%) of the subjects retuned the FIT-kits, showing high compliance. Among the 129 subjects with positive FIT results, 92 (71.3%) underwent colonoscopy. Six cases (6.5%) of CRC were found. Based on the data collected, the level of awareness of stool examination and knowledge about CRC was poor amongst the participants. Gender, age group, ethnicity and risk factors (i.e. smoking, lack of exercise and low consumption of fresh fruits) were associated with positive FIT-kit results. In conclusion, CRC screening can be performed in the community with a single FIT-kit. Although CRC knowledge and awareness is poor in low-income communities, the average return rate of the FIT kits and rate of colonoscopy examination were 91.2% and 70.3%, respectively.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.5489-5494
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• 2013

In the current study we aimed to show the common YMDD motif mutations in viral polymerase gene in chronic hepatitis B patients during lamivudine and adefovir therapy. Forty-one serum samples obtained from chronic hepatitis B patients (24 male, 17 female; age range: 34-68 years) were included in the study. HBV-DNA was extracted from the peripheral blood of the patients using an extraction kit (Invisorb, Instant Spin DNA/RNA Virus Mini Kit, Germany). A line probe assay and direct sequencing analyses (INNO-LIPA HBV DR v2; INNOGENETICS N.V, Ghent, Belgium) were applied to determine target mutations of the viral polymerase gene in positive HBV-DNA samples. A total of 41 mutations located in 21 different codons were detected in the current results. In 17 (41.5%) patients various point mutations were detected leading to lamivudin, adefovir and/or combined drug resistance. Wild polymerase gene profiles were detected in 24 (58.5%) HBV positive patients of the current cohort. Eight of the 17 samples (19.5%) having rtM204V/I/A missense transition and/or transversion point mutations and resistance to lamivudin. Six of the the mutated samples (14.6%) having rtL180M missense transversion mutation and resistance to combined adefovir and lamivudin. Three of the mutated samples (7.5%) having rtG215H by the double base substituation and resistance to adefovir. Three of the mutated samples (7.5%) having codon rtL181W due to the missense transversion point mutations and showed resistance to combined adefovir and lamivudin. Unreported novel point mutations were detected in the different codons of polymerase gene region in the current HBV positive cohort fromTurkish population. The current results provide evidence that rtL180M and rtM204V/I/A mutations of HBV-DNA may be associated with a poor antiviral response and HBV chronicity during conventional therapy in Turkish patients.