• Journal of Mushroom
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• v.9 no.2
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• pp.53-58
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• 2011

In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3'- and 5'-RACE PCR and RT-PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.295-302
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• 2002

Uterine cells carry out proliferation and differentiation for preparation the embryonic implantation during pregnancy. Therefore regulation of the cell proliferation is an essential step for uterine preparation, but there is not much information about the proliferation related genes in pregnant uterus. To identify these implantation specific genes, a PCR-select cDNA subtraction method was employed and got a few genes. One of the identified genes is a novel gene encoding oral tumor suppressor doc-1. To detect the doc-1 expression on the pregnant uterus, dot blotting, RT-PCR, and in situ hybridization were employed. Dot blotting revealed that doc-1 mRNA expression increase after implantation. During normal pregnancy, doc-1 mRNA expression was detected as early as day 1 of pregnancy with RT-PCR. Its expression was increased about 15 times after embryonic implantation. doc-1 transcript was localized in luminal epithelial cells but it was very faint during preimplantation. After starting the implantation, it localized in the stromal cells; heightened expression of doc-1 correlates with intense stromal cell proliferation surrounding the implanting blastocyst on day 6 morning. However in the decidualized cells, the intensity of localized doc-1 mRNA was weak. From those results, it is revealed that doc-1 express at pregnant uterus of the mouse. In addition it is suggested that doc-1 is the gene regulating the proliferation of the luminal epithelial cells and stromal cells during early implantation and decidualization.

• Korean Journal of Veterinary Service
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• v.31 no.1
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• pp.71-77
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• 2008

Today swine respiratory disease is one of the most important diseases because of its economic losses and severe infection nationwide, and swine society as well as veterinary service are trying to prevent the diseases in Korea. This study would like to obtain some information useful for the control of the diseases. A total of 174 lung specimens with lesion consisted of 3 sorts; 60 were collected from nursey pigs requested for diagnostic service from March of 2006 to October of 2007, 58 finishing pigs and 56 sows were selected from slaughterhouse from September to November 2007. In the detection test of pathogens by PCR, porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae were positive in 95.4%, 31.6%, and 20.1%, respectively. Double infection rate with PCV2 and PRRS was 30.4%, PCV2 and M hyopneumoniae was 19.5%, triple infection with PCV2, PRRS and M hyopneumoniae was 5.7%, respectively.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.440-448
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• 2021

Helicobacter pylori (H. pylori) establishes long-term infections associated with severe gastric diseases such as peptic ulceration and gastric cancer. Exposure to an antibacterial agent can help regulate the expression levels of its pathogenic genes. In this study, we analyzed the transcriptional changes in H. pylori genes induced by β-caryophyllene. We used next-generation sequencing (NGS) to analyze RNA expression changes, and reverse transcription-polymerase chain reaction (RT-PCR) was performed as required to verify the results. The NGS results showed that 30 out of 1,632 genes were expressed differentially by β-caryophyllene treatment. Eleven genes associated with DNA replication, virulence factors, and T4SS components were significantly downregulated. RT-PCR confirmed that treatment reduced the expression levels of 11 genes. RT-PCR showed the reduced expression of 11 genes (dnaE, dnaN, holB, gyrA, cagA, vacA, secA, flgE, virB2, virB4, and virB8) following β-caryophyllene treatment. These results suggest that β-caryophyllene can modulate various H. pylori pathogenic determinants and be a potential therapeutic agent for H. pylori infection.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.143.3-144
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• 2003

A Korean isolate of Pepper mild mottle virus (PMMoV-Kr) was isolated from a diseased pepper crop in Chunchon, Korea. The isolate was biologically purified on Nicoticaa tabacum cv. Xanthi-nc by successive single local transfer steps, and propagated on N. tabacum cv. Samsun. PMMoV-Kr could systemically infect on N. glauca, N. benthmiana, N. occidentalis and Lycopersicon esculentum, which is typical of known isolates of PMMoV. PMMoV-Kr belongs to the pathotype P1,2 based on pepper-tobamoviral indicator experiments; Capsicn chinone harboring L3 gene revealed resistant (necrotic local lesion on inoculated leaf, HR) whereas L+, L1 and L2 pepper plants expressed susceptible reactions of mosaic systemic symptoms for the isolate. To confirm the pathology and delineate symptom determinant of the isolate, full-length cDNAs of PMMoV-Kr were amplified by RT-PCR with a primer set corresponding to the 5'- and 3'-ends of PMMoV. The RT-PCR molecules amplified from genome RNA of the isolate was cloned into the pUC18 vector. Full-length cDNA clones constructed under the control of the T7 RNA promoter could be successfully transcribed to produce in vitro transcript RNA. Infectivity of the capped transcripts and its progeny virus was verified by Western blot and RT-PCR analyses.

• Proceedings of the National Institute of Ecology of the Republic of Korea
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• v.4 no.4
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• pp.154-158
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• 2023

The Asiatic black bear, Ursus thibetanus, is among the most threatened or endangered species in Asia. For its conservation and management, sex identification of U. thibetanus using non-invasive samples (e.g., hair and/or feces) is potentially valuable. In this study, a non-invasive molecular method for sex identification of U. thibetanus samples collected from various countries was first utilized, and it was based on polymerase chain reaction (PCR) amplification of the amelogenin gene via PCRs. Thirty-three bear DNA samples, extracted not only from blood (n=9) but also from hair (n=18) and feces (n=6), were used. We performed sex-specific PCR amplifications of the amelogenin gene using a primer set, SE47 and SE48. The primer set could successfully amplify a single X-specific band for females and both X- and Y-specific bands for males from all blood (100%) and hair (100%) samples. In addition, the primer set could distinguish the sex of bears in four out of a total of six fecal samples (approximately 67%). This study's findings suggest that this molecular method can be applied to sex identification of Asiatic black bears from various Asian regions using non-invasive samples, such as hair and feces.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.19
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• pp.8351-8359
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• 2014

Background: Recent epidemiological data have implicated human papilloma virus (HPV) infection in the pathogenesis of head and neck cancers, especially oropharyngeal cancers. Although, HPV has been detected in varied amounts in persons with oral dysplasia, leukoplakias and malignancies, its involvement in oral tongue carcinogenesis remains ambiguous. Materials and Methods: HPV DNA prevalence was assessed by PCR with formalin fixed paraffin embedded sections (n=167) of oral tongue squamous cell carcinoma patients and the physical status of the HPV16 DNA was assessed by qPCR. Immunohistochemistry was conducted for p16 evaluation. Results: We found the HPV prevalence in tongue cancers to be 51.2%, HPV 16 being present in 85.2% of the positive cases. A notable finding was a very poor concordance between HPV 16 DNA and p16 IHC findings (kappa<0.2). Further molecular classification of patients based on HPV16 DNA prevalence and p16 overexpression showed that patients with tumours showing p16 overexpression had increased hazard of death (HR=2.395; p=0.005) and disease recurrence (HR=2.581; p=0.002) irrespective of their HPV 16 DNA status. Conclusions: Our study has brought out several key facets which can potentially redefine our understanding of tongue cancer tumorigenesis. It has emphatically shown p16 overexpression to be a single important prognostic variable in defining a high risk group and depicting a poorer prognosis, thus highlighting the need for its routine assessment in tongue cancers. Another significant finding was a very poor concordance between p16 expression and HPV infection suggesting that p16 expression should possibly not be used as a surrogate marker for HPV infection in tongue cancers. Interestingly, the prognostic significance of p16 overexpression is different from that reported in oropharyngeal cancers. The mechanism of HPV independent p16 over expression in oral tongue cancers is possibly a distinct entity and needs to be further studied.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.237-244
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• 2011

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vitro models. RT-PCR and real time PCR analysis respectively revealed that LH had no effect on c-Ski mRNA expression in the cultured granulosa cells regardless of LH treatment. Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally. Moreover, expression of mRNA of Arkadia, an E3 ubiquitin ligases, in luteinizing granulosa cells in vivo was assessed by realtime-PCR. The levels of Arkadia mRNA expression were unchanged during follicular growth and postovulatory luteinization. These findings suggest that Ski protein level may be regulated during luteinization at translational and/or post-translational level but not by Arkadia.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1359-1367
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• 2005

The gene encoding Pyrobaculum arsenaticum DNA polymerase (Par DNA polymerase) was cloned and sequenced. The gene consists of 2,361 bp coding for a protein with 786 amino acid residues. The deduced amino acid sequence of Par DNA polymerase showed a high similarity to archaeal family B-type DNA polymerases (Group I), and contained all of the motifs conserved in the family B-type DNA polymerases for $3'{\rightarrow}5'$ exonuclease and polymerase activities. The Par DNA polymerase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RP. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $Hirap^{TM}$ Heparin HP column chromatographies. The optimum pH of the purified enzyme was 7.5. The enzyme activity was activated by divalent cations, and was inhibited by EDTA and monovalent cations. The half-life of the enzyme at $95^{\circ}C$ was 6 h. Par DNA polymerase possessed associated $3'{\rightarrow}5'$ proofreading exonuclease activity, which is consistent with its deduced amino acid sequence. PCR experiment with Par DNA polymerase showed an amplified product, indicating that this enzyme might be useful in DNA amplification and PCR-based applications.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.498-508
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• 2019

Bacillus species were isolated from iru, a traditional fermented condiment in Nigeria. Polyphasic approach was used to evaluate the phylogenetic relationship and strain sub-type of the isolated species. Additionally, the phylogenetic profiles of the species isolated from iru were compared with those of bacilli isolated from different continents. The phylogenetic diversity analysis was performed using the combination of 16S rRNA gene sequencing, ITS-PCR, ITS-PCR-RFLP, and M13 RAPD-PCR. The analysis revealed that Bacillus subtilis U170B and B. subtilis U146A isolated from iru were the closest relatives of strains belonging to the phylogeny of B. subtilis sensu stricto and were related to other bacilli isolated from different continents that had functional benefits. The two isolated species exhibited resistance to acidic pH (pH 2.0). The survival rates of B. subtilis U170B, B. subtilis U146A, and B. clausii UBBC-07 (commercial probiotic strain) cultured at pH 2.0 for 3 h were 33.45, 12.44, and 9.53%, respectively. The strains were highly tolerant to bile salts [0.3% (w/v)]. B. subtilis U170B exhibited the highest cell viability (43.45%) when cultured for 3 h in the presence of bile salts, followed by B. subtilis U146A (25%) and B. clausii UBBC-07 (18.94%). B. subtilis U170B and B. subtilis U146A did not exhibit haemolytic activity and were susceptible to different antibiotics. Additionally, these two strains exhibited weak antagonistic activity against B. cereus. The diverse wild strains of B. subtilis can be used as a safe multifunctional starter culture for the industrial production of condiments with health benefits.