• Clinical and Experimental Pediatrics
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• v.53 no.2
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• pp.178-183
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• 2010

Purpose : The aim of this study was to identify mutations associated with macrolide resistance in Mycoplasma pneumoniae (MP) and to establish a cultural method to determine antimicrobial susceptibility. Methods : Nasopharyngeal aspirates (NPAs) were collected from 62 children diagnosed with MP pneumonia by a serologic method or polymerase chain reaction. The 23S rRNA and L4 ribosomal protein genes of MP were amplified and sequenced. To identify mutations in these 2 genes, their nucleotide sequences were compared to those of the reference strain M129. MP cultivation was carried out for 32 (28 frozen and 5 refrigerated) NPAs and M129 strain using Chanock's glucose broth and agar plate in a 5% $CO_2$ incubator at $37^{\circ}C$ and examined at 2-3 day intervals for 6 weeks. Results : Among the 62 specimens, 17 had M144V mutations in ribosomal protein L4. The A2064G mutation was observed in 1 specimen; its 23S rRNA gene was successfully sequenced. Culture for MP was successful from the M129 strain and 2 of the 5 NPAs that were refrigerated for no longer than 3 days. However, MP did not grow from the 28 NPAs that were kept frozen at $-80^{\circ}C$ since 2003. Conclusion : We found the M144V mutation of L4 protein to be common and that of domain V of 23S rRNA gene was relatively rare among MP. Studies on the prevalence of macrolide-resistant MP and the relationship between the mutations of 23S rRNA gene and ribosomal protein L4 will aid in understanding the mechanism of macrolide resistance in MP.

• The Korean Journal of Mycology
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• v.39 no.1
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• pp.31-38
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• 2011

In this study, 81 commercial strains of Pleurotus species cultivated in South Korea were analyzed with randomly amplified polymorphic DNA (RAPD) technique. Sequence characterized amplified region (SCAR) markers were developed by designing from one RAPD polymorhic band specific to Suhan strain. The SCAR primer pair 'S-OPA13-1' amplified a 590-bp fragment in the varieties originated from Suhan strain. The Blast search of S-OPA13-1 showed high homology to the POMFBO1 P. ostreatus cDNA clone MFB02-A05 and Laccaria bicolor S238N-H82. The results showed that this SCAR marker can clearly distinguish Suhan strains from Pleurotus spp.

• Journal of Life Science
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• v.29 no.6
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• pp.653-661
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• 2019

The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method-which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics-is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of $R^2$>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ${\pm}20%$ of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stemloop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.

• Journal of Animal Science and Technology
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• v.52 no.4
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• pp.329-336
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• 2010

Myogenic satellite cells (MSCs) are mononuclear, multipotent progenitors of adult skeletal muscle possessing a capacity of forming adipocyte-like cells (ALC). To identify the skeletal muscle type-specific myogenic and adipogenic genes during MSCs differentiation, total RNA was extracted from bovine MSCs, myotube-formed cell (MFC), and ALC from each of Beef shank, Longissimus dorsi, Deep pectoral, and Semitendinosus. DNA microarray analysis (24,000 oligo chip) comparing MSCs with MFC and ALC, respectively, revealed 135 differentially expressed genes (> 4 fold) among four cuts. Real-time PCR confirmed expression of 29 genes. Furthermore, the whole tissue sample RNAs analysis showed 6 differentially expressed genes in Beef shank. Among which, 1 gene in MSCs, 4 in MFC, and 1 in ALCs were highly expressed. This study will provide an insight for better understanding the molecular mechanism of differentiation of skeletal muscle type-specific MSCs. The identified genes may be used as marker to distinguish skeletal muscle types.

• Development and Reproduction
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• v.10 no.2
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• pp.97-103
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• 2006

Vitellogenin(Vg) is a sex specific serum protein present in sexually maturing female blood of oviparous vertebrates. Estrogen($E_2$) is a main inducer of hepatic Vg synthesis. We investigated the effects of androgen and growth hormone(GH) on regulation of Vg and estrogen receptor(ER) genes in Japanese eel. Immature eels($200{\sim}250\;g$) were given a single injection of $E_2(5{\sim}5,000\;{\mu}g/kg\;bw)$ alone, or in combination with eel recombinant GH(eGH, $1{\sim}10\;{\mu}g/kg$) or methyltestosterone(MT, $1{\sim}5\;mg/kg$) and sacrificed 10 days after the hormone treatments. Expression levels of ER and Vg genes from the liver were determined by means of reverse transcription and polymerase chain reaction(RT-PCR). Administration of $E_2$ stimulated Vg gene expression in a dose dependent manner. Levels of Vg mRNA after the injection of $E_2(500\;{\mu}g/kg)$ with MT(5mg/kg) or eGH($10\;{\mu}g/kg$) were much higher than in that of $E_2$ alone($500\;{\mu}g/kg$). Whereas, injection of either vehicle, eGH ($10\;{\mu}g/kg$) or MT(5mg/kg) alone did not induce the expression of Vg gene in the liver. ER mRNA was detected from the fish treated with vehicle alone. $E_2$ injection($5{\sim}500\;{\mu}g/kg\;bw$) increased this ER expression but dose dependent response was not clear. Addition of MT(5mg/kg) or eGH($10\;{\mu}g/kg$) did not affect $E_2-stimulated$ ER mRNA expression. This study confirms the necessity of $E_2$ as the primary factor for Vg gene expression and requirement of additional hormones such as MT or GH for the full expression of Vg mRNA, and suggests that the additive effect of MT or GH on Vg gene expression would be mediated by some unknown factors other than ER.

• Korean Journal of Microbiology
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• v.40 no.1
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• pp.1-6
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• 2004

Tetracycline resistant bacterial strains were isolated from 10 batches of Kimchi among 50 batches collected in Taegu restrict. The MIC of tetracycline ranged between 25 and> 100 ㎖/l. Total genomic DNA preparation from all 10 tetracycline resistant lactic acid bacterial isolates were subjected to PCR amplification with class-specific primers for tet(M) and tet(O). In only one isolate, HJ9, tet(M) was detected. By Southern blotting and hybridization with a tet(M)-specific probe, the tet(M) gene of HJ9 isolate could be localized on a plasmid. The partial nucleotide sequence and deduced amino acid sequence of tet(M) of HJ9 showed 90-99% and 94-100% homology to those of Gram positive bacteria, respectively. With sequencing of 16S rRNA, HJ9 isolate from Kimchi was identified as Lactobacillus sakei. From these results, Kimchi can be considered potential vehicle for the spread of antibiotic-resistant lactic acid bacteria along the food chain to the consumer.

• Development and Reproduction
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• v.15 no.2
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• pp.179-185
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• 2011

Some phytoestrogens in soy and red wine, for example, might have beneficiary rather than adverse effects. In particular, dietary soy intake seems to be highly correlated with protection of breast cancer, osteoporosis and cardiovascular disorders. However, questions persist on the potential adverse effects of the main soy constituent genistein (GS) on female reproductive physiology. Previously we found that prepubertal exposure to GS could activate the reproductive system of immature female rats leading to precocious puberty onset, and intracerebroventricularly (ICV) injected GS could directly activate hypothalamic kisspeptin-GnRH neuronal circuits in adult female rats. The present study was performed to examine the hypothalamus-specific GS effects in prepubertal female rats and which subtype of estrogen receptor is mediated in this GS effect. Prepubertal female rats (PND 30) were anaesthetized, treated with single dose of GS (3.4 ${\mu}g$/animal), and sacrificed at 2 hrs post-injection. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). ICV infusion of GS significantly lowered the transcriptional activities of mTOR (1:$0.361{\pm}0.058$ AU, p<0.001) but increased that of GAD67 (1:$1.285{\pm}0.099$ AU, p<0.05), which are known to act as an upstream modulator of kisspeptin and GnRH neuronal activities in the hypothalamus, respectively. GS administration enhanced significantly the mRNA levels of KiSS-1(1:$1.458{\pm}0.078$ AU, p<0.001), and exerted no effect on the mRNA level of kisspeptin receptor GPR-54 (1:$1.29{\pm}0.08$ AU). GnRH gene expression was significantly decreased in GS-treated group compared to control group (1:$0.379{\pm}0.196$ AU, p<0.05). There was no difference in the mRNA level of $ER{\alpha}$ in the GS-treated group compare to control group (1:$1.180{\pm}0.390$ AU, Fig. 3A). However, icv infusion of GS significantly increased the transcriptional activities of $ER{\beta}$ (1:$4.209{\pm}0.796$ AU, p<0.01, Fig. 3B). Taken together, the present study indicated that the acute exposure to GS could directly alter the hypothalamic GnRH modulating system in prepubertal female rats. Our study strongly suggested the involvement of $ER{\beta}$ pathway in GS's hypothalamus-specific action, and this idea is consistent with the GS's well-known $ER{\beta}$-mediated protective action in breast cancer.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Journal of Animal Science and Technology
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• v.44 no.1
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• pp.13-22
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• 2002

To identify the differentially expressed genes at growth stage of Hanwoo, we constructed the subtractive cDNA library from loin mRNA of 12- and 24-month old Hanwoo by PCR-based subtraction. The fourteen genes were confirmed by sequencing and reverse northern blot analysis, and they were selected as candidate of putative genes differentially expressed at the growth stage of Hanwoo. Three subtracted cDNA fragments that expressed specific signal to cDNA probe for 6-month-old loin of Hanwoo were highly homologous to those of the genes encoding EPV 20, Ca2＋ATPase, and TCTP, respectively. The nine cDNA clones showed intense signal to cDNA probe from 12-month-old loin of Hanwoo, and highly homologus to those of genes encoding VCP, HSP 70, aldolase A, MSSK1, GM-2 activator protein, ryanodine receptor, acidic ribosomal phosphoprotein p1, ADP/ATP translocase, and UCP 2, respectively. Two subtracted cDNA clones that expressed specific signal to cDNA probes for 12- and 24-month-old loin of Hanwoo were detected. One of them was highly homologus to the gene encoding ferrochelatase and the other was highly homologus to the gene encoding ADRP.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.3
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• pp.684-692
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• 2003

The herbal extract(YMT_02) is a modified extracts from Yukmijihwang-tang(YMJ) to promote memory-enhancing. The YMJ extracts has been widely used as replenishing yin and tonifying the kidneys herbal medicine for hundred years ia Asian countries. The purpose of this study is to: 1) quantitatively evaluate the memory-enhancing effect of YMT_02 by passive avoidance test, 2) statistical evaluation of candidate gene expression (pentraxin. PEP-19, transthyretin) in rat hippocampus. The hippocampi of YMT_02 and control group were dissected and mRNA was further purified. After synthesizing cDNA using oligo-dT primer, the cDNA were applied to Real Time PCR. The results were as follows : 1) passive avoidance test showed enhancing memory retentin by YMT_02 treatment, 2) expression of pentraxin, that accelerate degenerating of neuronal cell, was significantly decreased, 3) the mRNA of genes that has been known to be associated with protecting neuronal cell degeneration, such as PEP-19 and transthyretin, were significantly increased upon YMT_02 treatment. From above results, the administration of YMT_02 which tonify the function of Kidneys could enhance the ability of memory and learning. In addition, the administration of YMT_02 enhance memory retention through modulating particular gene (pentraxin, PEP-19, transthyretin) expressions in hippocampu.