• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.381-388
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• 2001

The present study was designed to obtain porcine growth hormone binding protein (pGHBP) improved biological activation as derived mutation in binding site with growth horlnone (GH). A 756 bp of fragment encoding the extracellular domain of pGHBP gene was cloned from the total RNA of porcine fat tissue by reverse transcriptase polymerase chain reaction (RT-PCR) and created mutation in positions 26 and 122 using site-directed mutagenesis method. Position 26 is one and it is near to get on five potential N-linked glycosylation sites located in the extracellular domain of porcine growth hormone receptor known to have a direct influence on combination with GH. Position 122 is known as one of conformational epitope in bovine. It was over-expressed in E. coli using pET-32(c) expression vector and precisely purified by S-protein agarose and enterokinase. In our results, we was obtained pmGHBP of 30 kDa. It suggests to study the effects of the pmGHBP on cell proliferation in vitro and growth rate in vivo after administration.

• Journal of Preventive Medicine and Public Health
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• v.38 no.3
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• pp.330-336
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• 2005

Objectives : The aim of this study was to investigate the effects of bisphenol A (BPA), an estrogen-like environmental endocrine disrupter, on the placental function and reproduction in rats. The mRNA levels of the placental prolactin-growth hormone(PRL-GH) gene family, placental trophoblast cell frequency and reproductive data were analyzed. Methods : The pregnancies of F344 Fisher rats ($160g{\pm}20g$) were detected by the presence of the copulatory plug or sperm in the vaginal smear, which marked Day 0 of pregnancy. Pregnant rats were divided into three groups. The control group was intraperitoneally injected with a sesame oil vehicle. The two remaining groups were injected with 50 or 500 mg/kg B.W/day of BPA, resuspended in sesame oil, on either days 7 to 11 or 16 to 20 of pregnancy, with the rats sacrificed on either day 11 or 20, respectively. The mRNA levels of PRL-GH and Pit-1a and b isotype genes were analyzed by Northern blot hybridization and reverse transcription-polymerase chain reaction. The hormone concentrations were analyzed by radioimmunoassay, and the frequency of the placental trophoblast cells observed by a histochemical study. Reproductive data, such as the placental weight and litter size, were surveyed on day 20. The fetal weight was surveyed for 4 weeks after birth. A statistical analysis was carried out using the SAS program (version 8.1). Results : The mRNA levels of the PRL-GH gene family, such as placental lactogen I, Iv and II, prolactin like protein A, C and Cv, and decidual prolactin-related protein were significantly reduced due to BPA exposure. The mRNA levels of the Pit-1a and b isotype genes, which induce the expression of the PRL-GH gene family in the rat placenta, were also reduced due to BPA exposure. The PL-Iv and PL-II concentrations were reduced in the BPA exposed group. During the middle to last stage of pregnancy (Days 11-20), a high dose of BPA exposure reduced the frequency of spongiotrophoblast cells, which are responsible for the secretion of the PRL-GH hormones. Reproductive data, such as the placental and fetal weights and the litter size, were reduced, but that of the pregnancy period was extended in the BPA exposed compared to the control group. Conclusions : BPA disrupts the placental functions in rats, which leads to reproductive disorders.

• Science of Emotion and Sensibility
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• v.3 no.2
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• pp.75-84
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• 2000

문명의 발달과 함께 수면부족으로 인한 여러 가지 스트레스와 질환이 증가하게 되어 최근 수면연구에 대한 관심이 증가하고 있다. 본 연구는 다양한 온열환경 조건에서의 쾌적한 수면을 위한 온열환경을 제시하기 위해, 5명의 여성 피험자를 대상으로 22$^{\circ}C$, 26$^{\circ}C$, 3$0^{\circ}C$의 일정온도 조건과 $25^{\circ}C$에서 1시간 후와 2시간 후에 각각 1, 2$^{\circ}C$를 상승시켜주는 변동온도 조건하에서 수면생리신호를 측정하였다. 그리고, 수면단계 평가를 이용하여 총 수면시간, 깊은 수면의 비율, 그리고 최초 수면시작 시간에서 최초의 서파 수면이 나타나기까지의 지연시간 등의 수면효율을 평가하였다. 그 결과, 일정온도 조건에서는 26$^{\circ}C$에서 총 수면시간(466.7$\pm$10.25분)과 깊은 수면의 비율(33.1$\pm$4.95%)은 타 조건에 비해 높게 나타났고, 최초 서파수면까지의 지연시간(9.8$\pm$3.33분)은 타 조건에 비해 낮게 나타나 쾌적한 수면을 위한 가장 적절한 온열조건임을 관찰할 수 있었다. 그리고 변동온도 조건에서는 4가지 온열조건간에는 큰 차이가 나타나지 않았지만, 모든 조건에서 일정온도 조건보다는 좋은 결과를 나타내었다. 또한 수면 중 신체 움직임과 설문 분석에서도 동일한 결과를 보였다. 본 연구를 통해, 수면생리신호를 이용한 수면 쾌적성 분석은 수면의 질적인 상태를 관찰하는데 매우 적합한 파라메터를 도출할 수 있으며, 여러 가지 수면환경 조건을 평가하는데 매우 유용한 지표가 될 수 있음을 보였다.e results suggest that hCG treatment at 7 days after insemination could be used to increase the pregnancy rate of embryo transfer, and transfer, and only the recipients with PUN concentration of <12 mg/dl were influenced by treatment with hCG./TEX>이었으며, 이는 화성 기원을 지시한다.sucrose를 이용한 2단계 희석이 수정란의 생존성을 향상시키는 것으로 나타났다. 또한 발육 단계별 생존성에 있어서는 발육이 진전된 확장배 반포 시에 동결하는 것이 배반포기에 동결하는 것 보다 유리한 것으로 나타났다.ody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다..ments of that period left both in Japan and Korea. "Hyojedo" in Korea is supposed to have been influenced by the letter design. Asite- is also considered to

• Korean Journal of Plant Resources
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• v.22 no.3
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• pp.273-280
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• 2009

A cDNA clone encoding a MDR-like ABC transporter protein was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (named as Brmdr 1; GenBank accession no.: DQ296184 ) had a total length of 4222 bp with an open reading frame of 3900 bp, and encoded a predicted polypeptide of 1300 amino acids with a molecular weight of 143.1 kDa. The BrMDR1 protein shared 71.0, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAN28720), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Brmdr1 was a low-copy gene. Expression pattern analysis revealed that Brmdr1 constitutively expressed in the root, stem petals and stamens, but with lower expression in leaves and open flowers. The domains analysis showed that BrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that BrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.

• BMB Reports
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• v.45 no.12
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• pp.748-753
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• 2012

A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3'-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus ($TMGS^+$). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and $TMGS^+$, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1525-1535
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• 2014

The xynC gene, which encodes high molecular weight xylanase from Paenibacillus sp. DG-22, was cloned and expressed in Escherichia coli, and its nucleotide sequence was determined. The xynC gene comprised a 4,419bp open reading frame encoding 1,472 amino acid residues, including a 27 amino acid signal sequence. Sequence analysis indicated that XynC is a multidomain enzyme composed of two family 4_9 carbohydrate-binding modules (CBMs), a catalytic domain of family 10 glycosyl hydrolases, a family 9 CBM, and three S-layer homologous domains. Recombinant XynC was purified to homogeneity by heat treatment, followed by Avicel affinity chromatography. SDS-PAGE and zymogram analysis of the purified enzyme identified three active truncated xylanase species. Protein sequencing of these truncated proteins showed that all had identical N-terminal sequences. In the protein characterization, recombinant XynC exhibited optimal activity at pH 6.5 and $65^{\circ}C$ and remained stable at neutral to alkaline pH (pH 6.0-10.0). The xylanase activity of recombinant XynC was strongly inhibited by 1 mM $Cu^{2+}$ and $Hg^{2+}$, whereas it was noticeably enhanced by 10 mM dithiothreitol. The enzyme exhibited strong activity towards xylans, including beechwood xylan and arabinoxylan, whereas it showed no cellulase activity. The hydrolyzed product patterns of birchwood xylan and xylooligosaccharides by thin-layer chromatography confirmed XynC as an endoxylanase.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2036-2045
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

• Journal of Bio-Environment Control
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• v.11 no.3
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• pp.139-143
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• 2002

Cotton Glutathione S-Transferase (GST: EC 2.5.1.18) was cloned and overexpressed in tobacco (Nicotiana tabacum) plants. Northern blot analysis confirmed the successful transformation of cotton gst gene in tobacco plant. Type I and Type ll transcript patterns were identified in transgenic tobacco plants and only Type I transcripts were discussed in this paper, The activity of GST in the type II transgenic plants was about 1.5-fold higher than those of the wild type and non-expresser by using 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione as the substrate. The expression of cotton GST in tobacco plants proved that Gh-5 could be translated into functional protein. Type II transgenic plants produced functional GST in the cells. The effects of cotton GST in the seedlings was evaluated by growing the control and transgenic seedlings at $15^{\circ}C$ in the growth chamber in the light. Overexpressors were grown well compared to the control plants (non-expressors). lo test far tolerance to salinity, seeds of Gh-5 overexpressors and the wild type Xanthi seedlings were grown at 0, 50, 100, 150, and 200 mM NaCl solution. Gh-5 transgenic seedlings showed higher growth rate over control seedlings on 50 and 100 mM NaCl solution. There was no difference in growth rate at 150 and 200mM NaCl concentration.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.236-244
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• 2014

The gene encoding ${\alpha}-\small{L}$-arabinofuranosidase (AFase) from Paenibacillus sp. DG-22 was cloned, sequenced, and expressed in Escherichia coli. The AFase gene (abfA) comprises a 1,509 bp open reading frame encoding 502 amino acids with a molecular mass of 56,520 daltons. The deduced amino acid sequence of the gene shows that AbfA is an enzyme consisting of only a catalytic domain, and that the enzyme has significant similarity to AFases classified into the family 51 of the glycosyl hydrolases. abfA was subcloned into the pQE60 expression vector to fuse it with a six-histidine tag and the recombinant AFase (rAbfA) was purified to homogeneity. The specific activity of the recombinant enzyme was 96.7 U/mg protein. Determination of the apparent molecular mass by gel-filtration chromatography indicated that AbfA has a tetrameric structure. The optimal pH and temperature of the enzyme were 6.0 and $60^{\circ}C$, respectively. The enzyme activity was completely inhibited by 1 mM $HgCl_2$. rAbfA was active only towards p-nitrophephenyl ${\alpha}-\small{L}$-arabinofuranoside and exhibited $K_m$ and $V_{max}$ values of 3.5 mM and 306.1 U/mg, respectively. rAbfA showed a synergistic effect in combination with endoxylanase on the degradation of oat spelt xylan and wheat arabinoxylan.