• BMB Reports
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• v.47 no.10
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• pp.563-568
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• 2014

Human genome projects have enabled whole genome mapping and improved our understanding of the genes in humans. However, many unknown genes remain to be functionally characterized. In this study, we characterized human chromosome 4 open reading frame 34 gene (hC4orf34). hC4orf34 was highly conserved from invertebrate to mammalian cells and ubiquitously expressed in the organs of mice, including the heart and brain. Interestingly, hC4orf34 is a novel ER-resident, type I transmembrane protein. Mutant analysis showed that the transmembrane domain (TMD) of hC4orf34 was involved in ER retention. Overall, our results indicate that hC4orf34 is an ER-resident type I transmembrane protein, and might play a role in ER functions including $Ca^{2+}$ homeostasis and ER stress.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1197-1203
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• 2007

A genomic DNA library of the bacterium Paenibacillus sp. DG-22 was constructed and the ${\beta}-xylosi-dase-positive$ clones were identified using the fluorogenic substrate $4-methylumbelliferyl-{\beta}-D-xylopyr-anoside$ $({\beta}MUX)$. A recombinant plasmid was isolated from the clone and 4.3-kb inserted DNA was sequenced. The ${\beta}-xylosidase$ gene (xylA) was comprised of a 2,106 bp open reading frame (ORF) en-coding 701 amino acids with a molecular weight of 78,710 dalton and a pI of 5.0. The deduced amino acid sequence of the xylA gene product had significant similarity with ${\beta}-xylosidases$ classified into family 52 of glycosyl hydrolases. The xylA gene was subcloned into the pQE60 expression vector to fuse with six histidine-tag. The recombinant ${\beta}-xylosidase$ $(XylA-H_6)$ was purified to homogeneity by heat-treatment and immobilized metal affinity chromatography. The pH and temperature optima of the $XylA-H_6$ enzyme were pH 5.5-6.0 and $60^{\circ}C$, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.4
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• pp.576-584
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• 2002

Bovine PAS-4 is an integral membrane glycoprotein expressed in mammary epithelial cells. Complementary DNA (cDNA) cloning of PAS-4 was performed by reverse-transcriptase polymerase chain reaction (RT-PCR) with oligonucleotide probes based on it's amino terminal and internal tryptic-peptides. The cloned PAS-4 cDNA was 1,852 nucleotides (nt) long and its open reading frame (ORF) was encoded 1,413 base long. The deduced amino acid sequence indicated that PAS-4 consisted of 471 amino acid residues with molecular weight of 52,796, bearing 8 potential N-glycosylation sites and 9 cysteine residues. Partial bovine CD36 cDNA from liver also was sequenced and the homology of both nucleotide sequence was 94%. Most of the identical amino acid residues were in the luminal/extracellular domains. Contrary to PAS-4, bovine liver CD36 displays 6 potential N-glycosylation sites, which were located, except for those at positions 101 and 171, at same positions as PAS-4 cDNA. Cysteine residues of PAS-4 and CD36 were same at position and in numbers. Northern blot analysis showed that PAS-4 was widely expressed, although its mRNA steady-state levels vary considerably among the analyzed cell types. PAS-4 possessed hydrophobic amino acid segments near the amino- and carboxyl-termini. Two short cytoplasmic tails of the amino- and carboxyl-terminal ends constituted of a 5-7 and 8-11 amino acid residues, respectively.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.113-118
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• 1999

The insulin-like growth factor (IGF) is found in all vertebrates and its type-II molecule is regarded as a fundamental embryonic growth factor during development. We have firstly identified, in this study, a cDNA clone corresponding to IGF-II (flIGF-II) from the adult brain of the teleost, Paralichthys olivaceus. We also examined the tissue expression of flIGF-II in several adult tissues by RT-PCR. The flIGF-II cDNA contained a complete ORF consisting of 215 amino acids and one stop codon. Its molecular characteristics appear to be similar to the previously identified IGF-II molecules, in which a common primary structure exhibiting B, C, A, D, and E domains is evidently observed. This cDNA clone seems to be cleaved at $Ala_{52}$ for the $NH_2$-end signal peptide and appears to produce a 98 amino acid-long E-peptide from the $Arg^{118}$. The functional B-D domain regions, therefore, include 65 amino acids and is able to encode a 7.4-kDa protein. The most prominent structural difference between IGF-I and IGF-II was that the D domain of IGF-II exhibits a two-codon-deleted pattern compared to the 8 amino acid-containing IGF-I. The insulin family signature in the A domain and six cysteins forming three disulfide bridges between the B and A domains were evolutionary-conserved from teleosts to mammalian IGF-II. Interestingly, the E-peptide region appears to provide a distinct hallmark between teleosts in amino acid composition. The flIGF-II shows 85.1% of sequence identity to salmon and trout, 90.6% to tilapia, and 98.4% to perch in amino acid level. In tissue expressions of IGF-II, it is very likely that flIGF-II has a significant expression in the adult brain. However, liver seems to be the main source for IGF-II production, and relatively low signals were observed in the adult muscle and kidney. Taken together, it would be concluded that the functional region for IGF-II mRNA is highly similar in phylogeny and is evolutionary, conserved as a mediator for the growth of vertebrates.

• Journal of Ginseng Research
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• v.42 no.4
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• pp.455-462
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• 2018

Background: Ginsenoside Rh2 has been known to enhance the activity of immune cells, as well as to inhibit the growth of tumor cells. Although the repertoire of genes regulated by Rh2 is well-known in many cancer cells, the epigenetic regulation has yet to be determined, especially for comprehensive approaches to detect methylation changes. Methods: The effect of Rh2 on genome-wide DNA methylation changes in breast cancer cells was examined by treating cultured MCF-7 with Rh2. Pyrosequencing analysis was carried out to measure the methylation level of a global methylation marker, LINE1. Genome-wide methylation analysis was carried out to identify epigenetically regulated genes and to elucidate the most prominent signaling pathway affected by Rh2. Apoptosis and proliferation were monitored to examine the cellular effect of Rh2. Results: LINE1 showed induction of hypomethylation at specific CpGs by 1.6-9.1% (p < 0.05). Genome-wide methylation analysis identified the "cell-mediated immune response"-related pathway as the top network. Cell proliferation of MCF-7 was retarded by Rh2 in a dose-dependent manner. Hypermethylated genes such as CASP1, INSL5, and OR52A1 showed downregulation in the Rh2-treated MCF-7, while hypomethylated genes such as CLINT1, ST3GAL4, and C1orf198 showed upregulation. Notably, a higher survival rate was associated with lower expression of INSL5 and OR52A1 in breast cancer patients, while with higher expression of CLINT1. Conclusion: The results indicate that Rh2 induces epigenetic methylation changes in genes involved in immune response and tumorigenesis, thereby contributing to enhanced immunogenicity and inhibiting the growth of cancer cells.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.45-51
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• 2014

A cecropin-A gene was isolated from the immunized larvae of the Japanese oak silkworm, Antheraea yamamai and designed Ay-CecA. The complete Ay-CecA cDNA consists of 419 nucleotides with 195 bp open reading frame encoding a 64 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propetide and a 37-residue mature peptide with a theoretical mass of 4046.81. The deduced amino acid sequence of the peptide evidenced a significant degree of identity (62 ~ 78% identity) with other lepidopteran cecropins. Like many insect cecropin, Ay-CecA also harbored a glycine residue for C-terminal amidation at the C-end, which suggests potential amidation. To understand this peptide better, we successfully expressed bioactive recombinant Ay-CecA in Escherichia coli that are highly sensitive to the mature peptide. For this, we fused mature Ay-CecA gene with insoluble protein ketosteroid isomerase (KSI) gene to avoid the cell death during induction. The fusion KSI-CecA protein was expressed as inclusion body. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC), and cleaved by cyanogen bromide (CNBr) to release recombinant Ay-CecA. The purified recombinant Ay-CecA showed considerably antibacterial activity against Gram-negative bacteria, E. cori ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. Our results proved that this peptide with a potent antibacterial activity may play a role in the immune response of Japanese oak silkworm.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.93-97
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• 2014

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl ${\beta}$-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.110-115
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• 2016

A small plasmid (pDK4) from the Antarctic marine organism Pseudoalteromonas sp. PAMC 21150, was purified, sequenced and analyzed. pDK4 was determined to be 3,480 bp in length with a G+C content of 41.64% and contains three open reading frames encoding a replication initiation protein (RepA), a conjugative mobilization protein (Mob) and a hypothetical protein. PCR-amplified pDK4 was cloned in high-copy pUC19 to yield the fusion vector pDOC153. The chloramphenicol resistance gene was inserted into pDOC153 to give an ampicillin and chloramphenicol-resistant, Pseudoalteromonas - Escherichia coli shuttle vector (7,216 bp; pDOC155). The TonB-dependent receptor (chi22718_IV ) and exochitinase (chi22718_III ) genes from Arctic marine P. issachenkonii PAMC 22718 were cloned into pDOC155 to produce pDOC158 and pDOC165, respectively. Both vector derivatives were transferred into plasmid-free Pseudoalteromonas sp. PAMC 22137 by the triparental mating method. PCR experiments showed that the genes were stably maintained both in Pseudoalteromonas sp. PAMC 22137 and E. coli $DH5{\alpha}$ cells, indicating the potential use of pDOC155 as a new gene transfer system into marine Pseudoalteromonas spp.