• Molecules and Cells
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• 제25권3호
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• pp.446-451
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• 2008

We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.

• 한국발생생물학회지:발생과생식
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• 제26권2호
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• pp.37-47
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• 2022

Photoperiod has well been established to regulate testicular activities in golden hamsters. These animals breed actively around summer but become infertile in winter. In males, testicles are full of multistep germ cells including spermatozoa in summer. But in winter only fundamental cells consisting of the testicles are detected. The testicular degeneration is accompanied by the reduced levels of blood gonadotropins and testosterone. In this study, the expressions of gonadotropin subunit genes were investigated in the reproductive active and inactive testicles. And parts of sequences of the gonadotropin subunits were identified and compared with those of other rodents. As results, common gonadotropin alpha (CGa), follicle-stimulating hormone (FSH) β, and luteinizing hormone (LH) β genes were equivalently detected in pituitaries of both sexually active and inactive animals. In considering low concentrations of gonadotropin hormones determined in pituitary, the present findings imply that the processes involved in translation and/or formation of functional hormones could be impeded in the sexually inactive hamsters. All the nucleotide sequences of gonadotropin subunits identified in this study were same as those reported previously except for one base in CGa. An unsure amino acid deduced from the CGa sequence was confirmed from mRNA sequencing. The outcomes mentioned above suggest that animals with regressed testes prepare for the sexually active period forthcoming in the future.

• 한국균학회지
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• 제50권3호
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• pp.195-204
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• 2022

A fungal strain, designated KNUF-20-NI009, was isolated from soil collected from Gunsan-si, Jeollabuk-do, Korea. The isolate showed cultural features typical of the genus Xenoroussoella. Colonies cultivated on malt extract agar were olivaceous-brown to pale olivaceous-white at the margins, with undersides of dark olivaceous to olivaceous-brown and a white margin. The conidia, with a size range of 2.7-5.1×1.6-3.3 ㎛ ($\bar{x}=3.6\times2.6{\mu}m$, n=50), were globoid to ellipsoid in shape, hyaline when immature, becoming light brown to golden-brown when mature, and characterized by 1 or 2 guttules. Multi-locus sequence analysis based on a combined dataset of internal transcribed spacer regions (ITS), large subunit rDNA (LSU), small subunit rDNA (SSU), translation elongation factor 1-alpha (TEF1α), and RNA polymerase II largest subunit (RPB2) sequences revealed KNUF-20-NI009 to be a strain of Xenoroussoella triseptata. This is the first report of this species in Korea.

• BMB Reports
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• 제44권8호
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• pp.529-534
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• 2011

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.

• Molecules and Cells
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• 제41권6호
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• pp.523-531
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• 2018

Tumour metastasis is one of the most serious challenges of cancer as it is the major cause of mortality in patients with solid tumours, including osteosarcoma (OS). In this regard, anti-metastatic genes have potential for metastasis inhibition strategies. Recent evidence showed the importance of breast cancer metastasis suppressor 1 (BRMS1) in control of OS invasiveness, but the regulation of BRMS1 in OS remains largely unknown. Here, we used bioinformatics analyses to predict BRMS1-targeting microRNAs (miRNAs), and the functional binding of miRNAs to BRMS1 mRNA was evaluated using a dual luciferase reporter assay. Among all BRMS1-targeting miRNAs, only miR-151b, miR-7-5p and miR-3200-5p showed significant expression in OS specimens. Specifically, we found that only miR-3200-5p significantly inhibited protein translation of BRMS1 via pairing to the 3'-UTR of the BRMS1 mRNA. Moreover, we detected significantly lower BRMS1 and significantly higher miR-3200-5p in the OS specimens compared to the paired adjacent non-tumour bone tissues. Furthermore, BRMS1 and miR-3200-5p levels were inversely correlated to each other. Low BRMS1 was correlated with metastasis and poor patient survival. In vitro, overexpression of miR-3200-5p significantly decreased BRMS1 levels and promoted OS cell invasion and migration, while depletion of miR-3200-5p significantly increased BRMS1 levels and inhibited OS cell invasion and migration. Thus, our study revealed that miR-3200-5p may be a critical regulator of OS cell invasiveness.

• Journal of Animal Science and Technology
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• 제46권4호
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• pp.563-570
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• 2004

Insulin-like growth factors(IGF)s-I과 -II ligands와 이들의 receptors 및 6종류의 IGF-binding proteins(IGFBPs)로 구성된 IGF systim은 여러 종류의 세포의 생존, 증식 및 분화에 있어서 매우 중요한 역할을 한다. 리튬은 in vitro에서 여러 종류의 세포의 생존과 증식의 조절제로 알려져 있다. 본 연구는 IGF-I, IGF-I receptor 및 IGF carrier로서 주로 IGFBP-3를 발현하는 rat C6 glioma cell에서 LiCl로 유도된 세포 생존 및 증식의 변화와 IGF system components의 발현간의 관계를 구명하고자 수행되었다. 10%혈청을 함유하는 배양액에서 0, 2mM, 혹은 5mM LiCl을 첨가하여 C6 cell을 24시간 배양했을 시 세포의 생존률과 세포 수는 리튬 첨가에 의해 영향을 받지 않았다. 그러나 72시간 배양 했을 때 C6 cell은 명백히 리튬의 첨가수준에 따라 반응하였다. 0, 2m, 5mM LiCl 첨가수준에서 72시간 배양한 C6 cell은 각각 전형적인 세포분열, 세포분열 중지 및 세포사멸 양상을 보였다. 더욱이 사멸돼가는 세포는 reverse transcription-polymerase chain reaction으로 조사한 IGF-I, IGF receptor 및 IGFBP-3의 발현수준이 저하되었다. 흥미롭게도 혈청을 첨가하지 않은 배양조건 하에서 IGFBP-3에 대한 antisense oligodeoxyribonucleotide를 10${\mu}M$ 수준의로 첨가하여 배양했을 때도 표적 mRNA는 물론 세포 수도 줄었다. 종합하자면, C6 cell에서 리튬의 독성 효과의 일부는 이 제제에 의한 IGF system components의 발현 억제 효과에 의해 매개될 소지가 크다. 이러한 관점에서 IGRBP-3는 적어도 이 세포의 정상적인 증식을 위해 꼭 필요한(‘prrmissive') 역할을 할 수 있다는 점을 시사한다.

• Journal of Microbiology and Biotechnology
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• 제30권9호
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• pp.1420-1429
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• 2020

Corynebacterium glutamicum, an important industrial strain, has a relatively slower reproduction rate. To acquire a growth-boosted C. glutamicum, a descendant strain was isolated from a continuous culture after 600 generations. The isolated descendant C. glutamicum, JH41 strain, was able to double 58% faster (t_d=1.15 h) than the parental type strain (PT, t_d=1.82 h). To understand the factors boosting reproduction, the transcriptomes of JH41 and PT strains were compared. The mRNAs involved in respiration and TCA cycle were upregulated. The intracellular ATP of the JH41 strain was 50% greater than the PT strain. The upregulation of NCgl1610 operon (a putative dyp-type heme peroxidase, a putative copper chaperone, and a putative copper importer) that presumed to role in the assembly and redox control of cytochrome c oxidase was found in the JH41 transcriptome. Plasmid-driven expression of the operon enabled the PT strain to double 19% faster (t_d=1.82 h) than its control (t_d=2.17 h) with 14% greater activity of cytochrome c oxidase and 27% greater intracellular ATP under the oxidative stress conditions. Upregulations of genes those might enhance translation fitness were also found in the JH41 transcriptome. Plasmid-driven expressions of NCgl0171 (encoding a cold-shock protein) and NCgl2435 (encoding a putative peptidyl-tRNA hydrolase) enabled the PT to double 22% and 32% faster than its control, respectively (empty vector: t_d=1.93 h, CspA: t_d=1.58 h, and Pth: t_d=1.44 h). Based on the results, the factors boosting growth rate in C. gluctamicum were further discussed in the viewpoints of cellular energy state, oxidative stress management, and translation.

• Journal of Animal Science and Technology
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• 제64권3호
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• pp.481-499
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• 2022

This study aims to determine the effects of D-methionine (D-Met) isomer and the methionine precursor 2-hydroxy-4-methylthiobutanoic acid i (HMBi) supplementation on milk protein synthesis on immortalized bovine mammary epithelial cell (MAC-T). MAC-T cells were seeded using 10-cm dishes and cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) basic medium. The basic medium of DMEM/F12 was replaced with the lactogenic DMEM/ F12 differentiation medium when 90% of MAC-T cells reached confluency. The best dosage at 0.6 mM of D-Met and HMBi and incubation time at 72 h were used uniformly for all treatments. Each treatment was replicated six times wherein treatments were randomly assigned in a 6-well plate. Cell, medium, and total protein were determined using a bicinchoninic acid protein assay kit. Genes, proteomics and metabolomics analyses were also done to determine the mechanism of the milk protein synthesis pathway. Data were analyzed by two-way analysis of variance (ANOVA) with supplement type and plate as fixed effects. The least significant difference test was used to evaluate the differences among treatments. The HMBi treatment group had the highest beta-casein and S6 kinase beta-1 (S6K1) mRNA gene expression levels. HMBi and D-Met treatments have higher gene expressions compared to the control group. In terms of medium protein content, HMBi had a higher medium protein quantity than the control although not significantly different from the D-Met group. HMBi supplementation stimulated the production of eukaryotic translation initiation factor 3 subunit protein essential for protein translation initiation resulting in higher medium protein synthesis in the HMBi group than in the control group. The protein pathway analysis results showed that the D-Met group stimulated fructose-galactose metabolism, glycolysis pathway, phosphoinositide 3 kinase, and pyruvate metabolism. The HMBi group stimulated the pentose phosphate and glycolysis pathways. Metabolite analysis revealed that the D-Met treatment group increased seven metabolites and decreased uridine monophosphate (UMP) production. HMBi supplementation increased the production of three metabolites and decreased UMP and N-acetyl-L-glutamate production. Taken together, D-Met and HMBi supplementation are effective in stimulating milk protein synthesis in MAC-T cells by genes, proteins, and metabolites stimulation linked to milk protein synthesis.

• 식물병연구
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• 제25권2호
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• pp.94-97
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• 2019

육묘장에서 파프리카 유묘의 줄기와 뿌리가 갈변하고 잎이 시드는 증상이 나타났으며 병든 어린 식물체는 묘판에 서있지 못하고 쓰러졌다. 파프리카 줄기와 뿌리의 감염 부위에서 곰팡이 단포자 균주를 획득하였다. 분리 균주를 carnation leaf agar에 배양하여 균학적 특성을 관찰한 결과, 대형분생포자는 약간 굽은 낫 모양으로 무색이고 $18-26{\times}3-5{\mu}m$이며 1-3개의 격벽이 있었다. 소형분생포자는 타원형으로 무색이고 $6-10{\times}2-4{\mu}m$이며 격벽이 없었다. 후벽포자는 원형으로 $7-12{\mu}m$이며 균사의 끝이나 중간에 형성되었다. 균주의 포자현탁액을 파프리카 종자와 유묘에 접종하여 병원성을 검정한 결과, 이 병원균은 종자의 발아를 억제하는 영향을 주었으며 유묘에는 잘록병 증상이 나타났다. 병든 파프리카 유묘에서 분리한 균주의 형태적 특성을 비롯하여 TEF 및 RPB1 유전자 염기서열 분석을 통해 원인균을 Fusarium oxysporum으로 동정하였으며, 파프리카 잘록병으로 명명하여 국내 처음으로 보고하고자 한다.

• 대한한의학방제학회지
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• 제26권4호
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• pp.307-318
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• 2018

Objectives : Endoplasmic reticulum (ER) stress designates cellular responses to the accumulation of misfolded and unfolded proteins in ER, which is related to a variety of liver diseases. Present study investigated the inhibitory effects of Litsea japonica flesh water extract (LJE) aganist ER stress. Methods : After HepG2 cells were pretreated with LJE and subsequently exposed to tunicamycin (Tm) or thapsigargin (Tg), expression of C/EBP homologous protein (CHOP), glucose regulated protein 78 kDa (GRP78), asparagine synthetase (ASNS), and endoplasmic reticulum DnaJ homologue 4 (ERDJ4) were determined by immunoblot and real-time PCR analysis. Three canonical signaling pathways in response to ER stress were examined to explore molecular mechanisms involved. Results : Pretreatment of 1 mg/mL LJE inhibited Tm- or Tg-induced CHOP expression, while L. japonica fruit water extract did not. In addition, LJE decreased the levels of GRP78, ASNS, and ERDJ4 mRNA by Tm. Moreover, phosphorylations of eukaryotic translation initiation factor $2{\alpha}$ and inositol-requiring enzyme 1, expression of nuclear form of activating transcription factor $6{\alpha}$, and transactivation of ER stress response element- and unfolded protein response element-harboring luciferase activities were inhibited by LJE pretreatment. Conclusions : Present results suggest that LJE would be a candidate to prevent or treat ER stress-mediated liver injuries.