• IMMUNE NETWORK
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• v.3 no.3
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• pp.219-226
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• 2003

Background: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than $10^9$ members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to $10^{14}$ molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. Methods: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. Results: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. Conclusion: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

• Molecules and Cells
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• v.26 no.1
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• pp.26-33
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• 2008

The plasmid pJB01, a member of the pMV158 family isolated from Enterococcus faecium JC1, contains three open reading frames, copA, repB, and repC. Plasmids included in this family produce counter-transcribed RNA (ctRNA) that contributes to copy number control. The pJB01 ctRNA, a transcript which consists of 54 nucleotides (nts), is encoded on the opposite strand from the copA/repB intergenic region and partially overlaps an atypical ribosome binding site (ARBS) for repB. The ARBS is integrated by the two underlined conserved regions: 5'-TTTTTGTNNNNTAANNNNNNNNNATG-3', and the ctRNA is complementary only to the 5' conserved sequence 5'-TTTTTGT-3'. This complementary sequence is located at a distance from the terminal loop of the ctRNA secondary structure. The ctRNA structure predicted by the mfold program suggests the possible generation of a terminal and an internal hairpin loop. The amount of in vitro translation product of repB mRNA was inversely proportional to the ctRNA concentration. Mutations in the terminal and internal hairpin loops of the ctRNA had inhibitory effects on its binding to the target mRNA. We propose that the intact structures of the terminal and internal hairpin loops, respectively, play important roles in forming the initial kissing and extending complexes between the ctRNA and target mRNA and that these regulate the copy number of this plasmid.

• Molecules and Cells
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• v.45 no.7
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• pp.435-443
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• 2022

In response to environmental changes, signaling pathways rewire gene expression programs through transcription factors. Epigenetic modification of the transcribed RNA can be another layer of gene expression regulation. N⁶-adenosine methylation (m⁶A) is one of the most common modifications on mRNA. It is a reversible chemical mark catalyzed by the enzymes that deposit and remove methyl groups. m⁶A recruits effector proteins that determine the fate of mRNAs through changes in splicing, cellular localization, stability, and translation efficiency. Emerging evidence shows that key signal transduction pathways including TGFβ (transforming growth factor-β), ERK (extracellular signal-regulated kinase), and mTORC1 (mechanistic target of rapamycin complex 1) regulate downstream gene expression through m⁶A processing. Conversely, m⁶A can modulate the activity of signal transduction networks via m⁶A modification of signaling pathway genes or by acting as a ligand for receptors. In this review, we discuss the current understanding of the crosstalk between m⁶A and signaling pathways and its implication for biological systems.

• Journal of Life Science
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• v.25 no.10
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• pp.1184-1195
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• 2015

The zebrafish is an emerging vertebrate model organism in reproductive biology. The oocyte maturation of zebrafish is triggered by maturation inducing hormone (MIH, 17α,20β-Dihydroxy-4-pregnen-3-one). In almost all animals, the oocyte maturation is governed by activation of pre-MPF which consists of cyclinB and inactive Cdk1. In the oocyte of Xenopus and mice, the activity of Cdk1 is regulated in two ways, one is the interaction with cyclinB and the other is phosphorylation/dephosphorylation of T14/Y15 residues on the Cdk1 by Wee1 and Cdc25. Unlike Xenopus and mice that have a sufficient amount of pre-MPF, pre-MPF is absent in GV oocyte of most teleost including zebrafish. Therefore, the activation of MPF during zebrafish oocyte maturation might totally depend on de novo synthesis of cyclinB proteins. It is reported that the translation of maternal mRNA is regulated by combination of several RNA binding proteins such as CPEB, Dazl, Pum1/Pum2, and insulin-like growth factor2 mRNA-binding protein 3 in the zebrafish oocytes. However, the definitive mechanism of these proteins to regulate the translation of stored maternal mRNAs remains to be elucidated. Therefore, the investigation of the maturation process of the zebrafish oocyte will provide new information that can help identify the role of translational control in early vertebrate oocyte maturation.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.584-590
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• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding ${\beta}-xylosidase$, a major xylanolytic enzyme, was previously cloned and sequenced by the present authors. Sequence analysis indicated that translation of the xylA gene was initiated from the noncanonical initiation codon UUG, confirmed by analyzing three different amber (UAG) mutants of the xylA gene. In the present study, the UUG initiation codon was mutated into AUG or GUG, and the effects of the mutations on the XylA synthesis were examined. The AUG initiation codon was found to direct the highest level of ${\beta}-xylosidase$ synthesis; three-fold and fourteen-fold more enzyme activity than the UUG codon in E. coli and B. subtilis cells, respectively. Surprisingly, contrary to other systems reported to date, the UUG start codon was found next to AUG in the relative order of translational efficiency in both organisms. In addition, a greater abundance of the xylA mRNA was detected with the AUG start codon in both of these host cells than with GUG or UUG. Northern blot and Toeprint assays revealed that this was due to enhanced stability of mRNA with the AUG initiation codon. As expected, the ${\beta}-xylosidase$ protein level in the bacterial cells containing mRNA with the AUC start codon was also much higher than the levels with the other two different mRNAs.

• Applied Biological Chemistry
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• v.32 no.3
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• pp.222-231
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• 1989

Three classes of proteinase inhibitors are known in soybean; the Kunitz trypsin inhibitor (SKTI), the Bowman-Birk proteinase inhibitor and its isoinhibitors. To study the molecular structure and expression characteristics of the SKTI, antibody was obtained by immunizing rabbit with the SKTI purified from soybean by preparative electrophoresis. Anti-SKTI antibody was not only specific for mature SKTI in soybean seed but also recognized the precursor which was synthesized in vitro. Translation in vitro was carried out in wheat germ extract with polyadenylated mRNA isolated from developing soybean seeds. One of the seed specific translation products, MW 24K, was identified to be the precursor for the SKTI by immunoprecipitation with anti-SKTI antibody. Mature SKTI of MW 20K, however, was not detected in the translates in vitro. These results suggest that the precursor polypeptide is synthesized from the mRNA and is cleaved to yield mature SKTI in soybean seed. The SKTI gene was expressed with the maturation of soybean seed in a tissue-specific and development stage-specific manner.

• Journal of Life Science
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• v.17 no.7 s.87
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• pp.1023-1025
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• 2007

Ajuba is a number of proteins containing cytosolic LIM domain. Its function may provide a new pathway whereby cell-cell adhesive events are transmitted to the nucleus to regulate cell proliferation and differentiation decisions. Here, Ajuba gene expression was investigated its molecular properties associated with endoplasmic reticulum (ER) stresses (tunicamycin, DTT, A23187 and BFA) which induced remarkable ex-pression of Ajuba mRNA. The mRNA half life of Ajuba was also determined, its half life of Ajuba mRNA in FRTL-5 cells was approximately 2 hr after the initial translation. Although the obvious bioligical function of Ajuba is not clear, on the base of the results, Ajuba gene expression is deeply associated with ER stresses.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.1
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• pp.133-140
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• 2005

Although amino acids are substrates for the synthesis of proteins and nitrogen-containing compounds, it has become more and more clear over the years that these nutrients are also extremely important as regulators of body protein turnover. The branched-chain amino acids (BCAAs) together or simply leucine alone stimulate protein synthesis and inhibit protein breakdown in skeletal muscle. However, it was only recently that the mechanism(s) involved in the regulation of protein turnover by BCAAs has begun to be defined. The acceleration of protein synthesis by these amino acids seems to occur at the level of peptide chain initiation. Oral administration of leucine to food-deprived rats enhances muscle protein synthesis, in part, through activation of the mRNA binding step of translation initiation. Despite our knowledge of the induction of protein synthesis by BCAAs, there are few studies on the suppression of protein degradation. The recent findings that oral administration of leucine rapidly reduced $N^{\tau}$-methylhistidine (3-methylhistidine; MeHis) release from isolated muscle, an index of myofibrillar protein degradation, indicate that leucine suppresses myofiblilar protein degradation. The details of the molecular mechanism by which leucine inhibits proteolysis is just beginning to be elucidated. The purpose of this report was to review the current understanding of how BCAAs act as regulators of protein turnover.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.267-273
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• 2008

Water-soluble fraction (WSF) from edible mushroom hinmogi (Tremella fuciformis) were obtained by water extraction, and polysaccharides in the WSF were separated by ethanol precipitation. The inhibitory effects of the polysaccharides on 3T3-L1 adipocyte differentiation were evaluated by the reduction of peroxisome proliferators-activated receptor ${\gamma}$ ($PPAR{\gamma}$) translation, triglyceride accumulation, Oil Red-O staining, and expression levels of $PPAR{\gamma}$, CCAAT/enhancer binding protein a (C/$EBP{\alpha}$), and leptin. The $PPAR{\gamma}$ translation in 3T3-L1 cells was inhibited by the treatment with polysaccharide precipitated by 80% ethanol (P80) which showed highest inhibitory activity among polysaccharides tested. In addition, treatment of P80 to 3T3-L1 cells significantly inhibited the triglyceride accumulation, Oil Red-O staining, and mRNA expression of $PPAR{\gamma}$, C/$EBP{\alpha}$, and leptin in a dose-dependent manner. Based upon these results, P80 from edible mushroom hinmogi shows the inhibitory activity on the differentiation of 3T3-L1 adipocytes. Therefore, it might be employed as a potential anti-obesity material.

• Journal of Integrative Natural Science
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• v.12 no.4
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• pp.127-132
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• 2019

Programmed cell death 4 (PDCD4) is a novel tumor suppressor that function in the nucleus and the cytoplasm and appears to be involved in the regulation of transcription and translation. Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate when cells exposed to environmental stresses. Since PDCD4 has implicated in translation repression through direct interaction with eukaryotic translation initiation factor 4A (eIF4A), we here investigated if PDCD4 has a functional role in the process of SG assembly under oxidative stresses. Using immunofluorescence microscopy, we found that PDCD4 is localized to SGs under oxidative stresses. Next, we tested if knockdown of PDCD4 has an effect on the assembly of SG using PDCD4-specific siRNA. Interestingly, SG assembly was accelerated and this effect was caused by sensitization of phosphorylation of eIF2α and dephosphorylation of eIF4E binding protein (4E-BP). These results suggest that PDCD4 has an effect on SG dynamics and possibly involved in cap-dependent translation repression under stress conditions.