• 생명과학회지
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• 제22권6호
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• pp.713-722
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• 2012

Landrace와 Berkshire의 longissimus dorsi muscle으로부터 단백질 발현양상의 차이를 보기 위하여 2-DE실험을 통하여 분석한 결과 Landrace 에서 특이적으로 발현 양이 증가한 단백질들은 serum albumin precursor, troponin T (TnT; slow skeletal muscle), myoglobin였다. Berkshire에서 특이적으로 발현 양이 증가한 단백질들은 heat shock 27 kDa protein 1, troponin T (fast skeletal muscle), muscle creatine kinase, phosphoglucomutase 1, triosephosphate isomerase (Tpi 1), adenylate kinase isoenzyme 1 (AK1)였다. Landrace의 longissimus dorsi muscle에서는 slow skeletal muscle과 연관된 단백질들이 발현된 반면에 Berkshire에서는 fast skeletal muscle, 물질대사경로, 에너지 생산과 관련된 단백질들이 발현되었다. Berkshire를 이용하여 성장단계별로 단백질 발현을 분석해 본 결과 growing Berkshire에서 발현이 증가한 단백질은 aldehyde dehydrogenase 1 family, member L1 (ALDHL1)와 muscle creatine kinase이고 finishing Berkshire에서 발현이 증가한 단백질은 heat shock 27 kDa protein 1, TnT (slow skeletal muscle), TnT (fast skeletal muscle), serum albumin precursor, PGM 1, AK 1, Tpi 1였다. 이 결과는 Finishing Berkshire의 등심에서는 growing Berkshire에 비교하여 골격근육, 에너지물질대사, 세포골격 등이 보다 활성화된 것으로 사료된다.

• Journal of Acupuncture Research
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• 제23권3호
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• pp.177-190
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• 2006

Objectives : It has long been known about the anticancer effect of GRR-HAS, however, it has not been systemically determined the differentially regulated genes by GRR-HAS in cancer cells. The purpose of this study is to screen the GRR-HAS mediated differentially expressed genes in cancer cells such as HepG2 hepatoma cell lines. Oligonucleotide microarray and proteomic approaches were employed to screen the differential expression genes. Methods : GRR~HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of GRR-HAS (0.1, 0.5, 1.5, 10, $20mg/m{\ell}$) for 24 h. Cell toxicity was tested by MTT assay. To screen the differentially expressed genes in cancer cells, cells were treated with $1.5mg/m{\ell}$ of GRR-HAS. For oligonucleotide microarray assay, total RNA was used for gene expression analysis using oligonucleotide genechip (Human genome Ul33 Plus 2.0., Affimatrix Co.). For proteomic analysis, total protein was analyzed by 2D gel electrophoresis and Q-TOF mass spectrometer. Results : It has no cytotoxic effects on both HepG2 cells in all concentrations(0.1, 0.5, 1.5, 10,$20mg/m{\ell}$). In oligonucleotide microarray assay, the number of more than twofold differentially regulated known genes was 320 with 6 up-regulated and 314 down-regulated genes in HepG2 cells. In proteomic analysis, three spots were identified by 2D-gel electrophoresis and Q-TOF analysis. One down -regulated protein was protein disulfide isomerase and up-regulated proteins were fatty acid binding protein 1 and 14-3-3 gan1lTIa protein by $1.5mg/m{\ell}$ of CRR-HAS. Discussion : This study showed the comprehensive gene expression analysis using oligonucleotide microarray for the screening of GRR-HAS mediated differentially regulated genes. These results will provide a better application of GRR-HAS in cancer field and drug target development.

• Asian-Australasian Journal of Animal Sciences
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• 제28권11호
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• pp.1573-1582
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• 2015

This study was to investigate the effect of soyabean isoflavones (SIF) on onset of puberty, serum hormone concentration, and gene expression in hypothalamus, pituitary and ovary of female Bama miniature pigs. Fifty five, 35-days old pigs were randomly assigned into 5 treatment groups consisting of 11 pigs per treatment. Results showed that dietary supplementation of varying dosage (0, 250, 500, and 1,250 mg/kg) of SIF induced puberty delay of the pigs with the age of puberty of pigs fed basal diet supplemented with 1,250 mg/kg SIF was significantly higher (p<0.05) compared to control. Supplementation of SIF or estradiol valerate (EV) reduced (p<0.05) serum gonadotrophin releasing hormone and luteinizing hormone concentration, but increased follicle-stimulating hormone concentration in pigs at 4 months of age. The expression of KiSS-1 metastasis-suppressor (KISS1), steroidogenic acute regulatory protein (StAR) and 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase ($3{\beta}-HSD$) was reduced (p<0.01) in SIF-supplemented groups. Expression of gonadotropin-releasing hormone receptor in the pituitary of miniature pigs was reduced (p<0.05) compared to the control when exposed to 250, 1,250 mg/kg SIF and EV. Pigs on 250 mg/kg SIF and EV also showed reduced (p<0.05) expression of cytochrome P450 19A1 compared to the control. Our results indicated that dietary supplementation of SIF induced puberty delay, which may be due to down-regulation of key genes that play vital roles in the synthesis of steroid hormones.

• 한국미생물·생명공학회지
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• 제35권4호
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• pp.272-277
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• 2007

P. pastoris는 대장균에 비해 정확한 접힘, 당화, 효율적인 분비기작등의 장점을 가지고 있어 재조합 단백질의 생산을 위한 균주로서 관심을 받고 있다. 또한 재조합 단백질의 효율적인 생산공정 개발을 위하여 배양공정 중 특정 시간이나 조건에서 발현될 수 있는 효율적인 유도성 프로모터의 개발도 중요 관심 분야이다. 본 연구에서는 연속배양을 이용하여 P. pastoris의 배양 중 특정 기질이 소모되었을 때 발현되는 자동 유도성 프로모터를 개발하기 위하여, 인산이 고갈되었을 때 과발현 되는 유전자들을 탐색하였다. 인산 제한 연속배양의 정상상태에서 얻어진 균체로 부터 total RNA와 mRNA를 분리하였고, 이로부터 cDN를 합성하여 인산제한조건에서 과발현되는 유전자들을 확보하였다. 그 중 빈도수가 높은 8종의 유전자 3-phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase(GAPDH), glucokinase, thiol-specific antioxidant protein, triosephosphate isomerase, sodium/phosphate symporter(NPS) 그리고 pyruvate decarboxylase를 선별하였고, Northern blot analysis를 수행한 결과 인산 섭취에 관련된 NPS 유전자가 인산제한조건에서 과발현 됨을 확인하였다. 본 연구실에서는 자동유도성 프로모터로서 NPS유래의 프로모터의 잠재성을 알아보기 위하여, 관련 유전자를 확보하여 외래 유전자를 이용한 발현연구를 진행 중이다.

• Journal of Forest and Environmental Science
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• 제30권2호
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• pp.243-252
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• 2014

Forest tree species usually takes for long periods to be harvested and cultivated but spearmints are a good model system for woody plant because of reducing and shortening cultivation time. Spearmints are good model plants (Mentha species) for research about terpenoids production and industrial essential oil manufacture. Isopentenyl pyrophosphate isomerase (Iso) and limonene synthase (Limo) are the key enzymes of terpenoid biosynthesis pathway. Transgenic and wild spearmints (Mentha spicata, MS) were cultured in vitro and assessed for the essential oil contents. The content of essential oil of transgenic spearmint also was enhanced slightly depending on the target terpenoid genes. In an attempt to increase productivity of terpenoids further, salt and fungal elicitation strategy was adopted on transgenic Mentha spicata. The salt (800 mM NaCl) as abiotic and two fungi (Botrytis cinerea and Glomerella cingulata) as biotic were used for elicitors. In the absence of salt stress four terpenoids were detected from the spearmint extracts, all of them being monoterpenes. On the other hand, the transgenic (MSIso) extracts contained eleven terpenoids (10 monoterpenes and 1 phenylpropene) while transgenic (MSLimo) extracts contained seven monoterpenes. After 3 days of fungal infection, the resistance indices further increased to 4.38, 3.89 and 2.04 for wild type, MSIso and MSLimo, respectively. The salt and fungal elicitators proved beneficial towards modifying both the terpenoids profile and improvement in the composition of essential oil. These results have important applications for the large-scale production of essential oils and forest biotechnology with respect to spearmint.

• KSBB Journal
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• 제26권6호
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• pp.517-522
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• 2011

In our previous study, we isolated and characterized a 1-deoxynojirimycin (DNJ)-producing bacterium, Bacillus subtilis MORI, from chungkookjang, a Korean traditional food. B. subtilis MORI was subjected to ${\gamma}$-irradiation and the resulting bacteria were screened for increased DNJ production. A mutant was identified that produced 7.6 times more DNJ and named B. subtilis MORI 3K-85. In this study, the protein profiles of both strains were compared by one-dimensional and two-dimensional gel electrophoresis (1-DE and 2-DE, respectively) under both native and denaturing conditions. The 1-DE native-PAGE and 1-DE SDS-PAGE analyses identified 5 and 7 bands, respectively, that were found at higher concentrations in B. subtilis MORI 3K-85 than in B. subtilis MORI. Similarly, 2-DE analyses identified 20 protein spots which were found at higher concentrations in B. subtilis MORI 3K-85. The peptide mass profiles of these 20 proteins were analyzed by MALDI-TOF and compared with peptide sequences of B. subtilis and B. amyloliquefaciens in the MASCOT database. This screening suggested that three dehydrogenases, an aldolase, a synthetase, an isomerase, a reductase, and a peroxidase are elevated in B. subtilis MORI 3K-85. Based on this data, one or more of the elevated 8 enzymes might be related to the DNJ biosynthetic pathway.

• Bulletin of the Korean Chemical Society
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• 제31권9호
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• pp.2497-2502
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• 2010

Arg13 is a conserved active-site residue in all known Pi class glutathione S-transferases (GSTs) and in most Alpha class GSTs. To evaluate its contribution to substrate binding and catalysis of this residue, three mutants (R13A, R13K, and R13L) were expressed in Escherichia coli and purified by GSH affinity chromatography. The substitutions of Arg13 significantly affected GSH-conjugation activity, while scarcely affecting glutathione peroxidase or steroid isomerase activities. Mutation of Arg13 into Ala largely reduced the GSH-conjugation activity by approximately 85 - 95%, whereas substitutions by Lys and Leu barely affected activity. These results suggest that, in the GSH-conjugation activity of hGST P1-1, the contribution of Arg13 toward catalytic activity is highly dependent on substrate specificities and the size of the side chain at position 13. From the kinetic parameters, introduction of larger side chains at position 13 results in stronger affinity (Leu > Lys, Arg > Ala) towards GSH. The substitutions of Arg13 with alanine and leucine significantly affected $k_{cat}$, whereas substitution with Lys was similar to that of the wild type, indicating the significance of a positively charged residue at position 13. From the plots of log ($k_{cat}/{K_m}^{CDNB}$) against pH, the $pK_a$ values of the thiol group of GSH bound in R13A, R13K, and R13L were estimated to be 1.8, 1.4, and 1.8 pK units higher than the $pK_a$ value of the wild-type enzyme, demonstrating the contribution of the Arg13 guanidinium group to the electrostatic field in the active site. From these results, we suggest that contribution of Arg13 in substrate binding is highly dependent on the nature of the electrophilic substrates, while in the catalytic mechanism, it stabilizes the GSH thiolate through hydrogen bonding.

• Asian-Australasian Journal of Animal Sciences
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• 제33권12호
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• pp.2021-2030
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• 2020

Objective: Quantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs. Methods: The cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins. Results: Based on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance. Conclusion: This study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

• Parasites, Hosts and Diseases
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• 제53권4호
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• pp.395-402
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• 2015

Non-human primates (NHPs) are confirmed as reservoirs of Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi. In this study, 197 fresh fecal samples from 8 NHP species in Qinling Mountains, northwestern China, were collected and examined using multilocus sequence typing (MLST) method. The results showed that 35 (17.8%) samples were positive for tested parasites, including Cryptosporidium spp. (3.0%), G. intestinalis (2.0%), and E. bieneusi (12.7%). Cryptosporidium spp. were detected in 6 fecal samples of Macaca mulatta, and were identified as C. parvum (n=1) and C. andersoni (n=5). Subtyping analysis showed Cryptosporidium spp. belonged to the C. andersoni MLST subtype (A4, A4, A4, and A1) and C. parvum 60 kDa glycoprotein (gp60) subtype IId A15G2R1. G. intestinalis assemblage E was detected in 3 M. mulatta and 1 Saimiri sciureus. Intra-variations were observed at the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) loci, with 3, 1, and 2 new subtypes found in respective locus. E. bieneusi was found in Cercopithecus neglectus (25.0%), Papio hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10%), and Rhinopithecus roxellana (9.5%), with 5 ribosomal internal transcribed spacer (ITS) genotypes: 2 known genotypes (D and BEB6) and 3 novel genotypes (MH, XH, and BSH). These findings indicated the presence of zoonotic potential of Cryptosporidium spp. and E. bieneusi in NHPs in Qinling Mountains. This is the first report of C. andersoni in NHPs. The present study provided basic information for control of cryptosporidiosis, giardiasis, and microsporidiosis in human and animals in this area.

• Molecules and Cells
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• 제28권4호
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• pp.383-388
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• 2009

The dehydration responsive element binding protein 2C (DREB2C) is a dehydration responsive element/C-repeat (DRE/CRT)-motif binding transcription factor that induced by mild heat stress. Previous experiments established that overexpression of DREB2C cDNA driven by the cauliflower mosaic virus 35S promoter (35S:DREB2C) resulted in increased heat tolerance in Arabidopsis. We first analyzed the proteomic profiles in wild-type and 35S:DREB2C plants at a normal temperature ($22^{\circ}C$), but could not detect any differences between the proteomes of wild-type and 35S: DREB2C plants. The transcript level of DREB2C in 35S: DREB2C plants after treatment with mild heat stress was increased more than two times compared with expression in 35S:DREB2C plants under unstressed condition. A proteomic approach was used to decipher the molecular mechanisms underlying thermotolerance in 35S:DREB2C Arabidopsis plants. Eleven protein spots were identified as being differentially regulated in 35S:DREB2C plants. Moreover, in silico motif analysis showed that peptidyl-prolyl isomerase ROC4, glutathione transferase 8, pyridoxal biosynthesis protein PDX1, and elongation factor Tu contained one or more DRE/CRT motifs. To our knowledge, this study is the first to identify possible targets of DREB2C transcription factors at the protein level. The proteomic results were in agreement with transcriptional data.