• KOREAN JOURNAL OF CROP SCIENCE
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• v.59 no.3
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• pp.385-389
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• 2014

Flavonols as a major kind of plant secondary metabolites are known for health-promoting compounds in onions (Allium cepa L.). The objectives of this study are to determine profiles of flavonol glycosides in different 75 onion accessions. A total of five flavonols (quercetin 3,4'-diglucoside, Q34'diG; quercetin 3-glucoside, Q3G; quercetin 4'-glucoside, Q4'G; isorhamnetin 4'-glucoside, I4'G; quercetin, Q) were identified from onion accessions. In positive ion mode using LC-ESI-MS, individual flavonols were confirmed from one and two glycosylation binding with aglycone such as quercetin and isorhamnetin. Total flavonol contents were distributed in white onion (range of 0.18-6.47 mg/g DW) and purple onion accessions (range of 2.39-6.47 mg/g), respectively. The mean of flavonol contents in purple onion (4.41 mg/g) showed 1.4-fold higher than white onion (3.23 mg/g). The Q34'diG and Q4'G were considered as the major compounds of flavonol glycosides in onion accessions.

• The Plant Pathology Journal
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• v.18 no.5
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• pp.251-258
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• 2002

A potyvirus was isolated from cultivated Iris plants showing leaf streak mosaic symptom. Reverse transcription and polymerase chain reaction (RT-PCR) product of 1 kb long which encoded partial nuclear inclusion B and N-terminal region of viral coat protein (CP) genes for potyviruses was successfully amplified with a set of potyvirus-specific degenerate primers with viral RNA samples from the infected leaves: The RT-PCR product was cloned into the plasmid vector and its nucleotide sequences were determined. The nucleotide sequence of a CDNA clone revealed that the virus was an isolate of Ornithogalum moseic virus (OrMV) based on BLAST search analysis and was denoted as OrMV Korean isolate (OrMV-Ky). To further characterize the CP gene of the virus, a pair of OrMV-specific primers was designed and used for amplification of the entire CP gene of OrMV-Kr, The virus was easily and reliably detected from virus-infected Iris leaves by using the RT-PCR with the set of virus-specific primers. The RT-PCR product of the CP gene of the virus was cloned and its sequences were determined from selected recombinant CDNA clones. Sequence analysis revealed that the CP of OrMV-Kr consisted of 762 nucleotides, which encoded 253 amino acid residues. The CP of OrMV-Ky has 94.1-98.0% amino acid sequence identities (20 amino acid alterations) with that of other three isolates of OrMV, Two NT rich potential N-glycosylation motif sequences, NCTS and NWTM, and a DAC triple box responsible for aphid transmission were conserved in CPs of all the strains of OrMV. The virus has 58.5-86.2% amino acid sequence identities with that of other 16 potyviruses, indicating OrMV to be a distinct species of the genus. OrMV-Ky was the most related with Pterostylia virus Yin the phylogenetic tree analysis of CP at the amino acid level. This is the first report on the occurrence of OrMV in Iris plants in Korea. Data in this study indicate that OrMV is found in cultivated Iris plants, and may have mixed infection of OrMV and Iris severe mosaic virus in Korea.

• Nuclear Medicine and Molecular Imaging
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• v.41 no.5
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• pp.343-349
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• 2007

Radioiodide transport has been extensively and successfully used in the evaluation and management of thyroid disease. The molecular characterization of the sodium/iodide symporter (NIS) and cloning of the NIS gene has led to the recent expansion of the use of radioiodide to cancers of the breast and other nonthyroidal tissues exogenously transduced with the NIS gene. More recently, discoveries regarding the functional analysis and regulatory processes of the NIS molecule are opening up exciting opportunities for new research and applications for NIS and radio iodide. The success of NIS based cancer therapy is dependent on achievement of maximal radioiodide transport sufficient to allow delivery of effective radiation doses. This in turn relies on high transcription rates of the NIS gene. However, newer discoveries indicate that nontranscriptional processes that regulate NIS trafficking to cell membrane are also critical determinants of radioiodide uptake. In this review, molecular mechanisms that underlie regulation of NIS transcription and stimuli that augment membrane trafficking and functional activation of NIS molecules will be discussed. A better understanding of how the expression and cell surface targeting of NIS proteins is controlled will hopefully aid in optimizing NIS gene based cancer treatment as well as NIS based reporter-gene imaging strategies.

• Animal cells and systems
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• v.4 no.2
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• pp.157-163
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• 2000

Yeast pheromone a-factor is a 13-amino acid peptide hormone that is synthesized as a part of a larger precursor, prepro-$\alpha$-factor, consisting of a signal peptide and a proregion of 64 amino acids. The carboxy-terminal half of the precursor contains four tandem copies of mature $\alpha$-factor. To investigate the molecular basis of intracellular sorting, proteolytic processing, and storage of the peptide hormone, yeast prepro-$\alpha$-factor precursors were heterologously expressed in rat pituitary $GH_3 cells. When cells harboring the precursor were metabolically labeled, a species of approximately 27 kD appeared inside the cells. Digestion with peptide: N-glycosidase F (PNG-F) shifted the molecular mass to a 19 kD, suggesting that the 27 kD protein was the glycosylated form as in yeast cells. The nascent polypeptide is efficiently targeted to the ER in the $GH_3 cells, where it undergoes cleavage of its signal peptide and core glycosylation to generate glycosylated pro-a-factor. To look at the post ER intracellular processing, the pulse-labelled cells were chased up to 2 hrs. The nascent propeptides disappeared from the cells at a half life of 30 min and only 10-25% of the newly synthesized, unprocessed precursors were stored intracellularly after the 2 h chase. However, about 20% of the pulse-labeled pro-$\alpha$-factor precursors were secreted into the medium in the pro-hormone form. With increasing chase time, the intracellular level of propeptide decreased, but the amount of secreted propeptide could not account for the disappearance of intracellular propeptide completely. This disappearance was insensitive to lysosomotropic agents, but was inhibited at $16^{circ}C or 20^{\circ}C$, suggesting that the turnover of the precursors was not occurring in the secretory pathway to trans Golgi network (TGN) or dependent on acidic compartments. From these results, it is concluded that a pan of these heterologous precursors may be processed at its paired dibasic sites by prohormone processing enzymes located in TGN/secretpry vesicles producing small peptides, and that the residual unprocessed precursors may be secreted into the medium rather than degraded intracellularly.

• Archives of Pharmacal Research
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• v.22 no.5
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• pp.485-490
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• 1999

The mechanism of action of fish oil (FO), currently used in different chronic inflammatory conditions such as rheumatoid arthritis (RA), is not completely understood, although it is thought that it could alter the metabolism of endogenous autacoids. In addition, we hypothesized that the known capability of fatty acids (FA) of stabilizing serum albumin and perhaps other proteins, may be of pharmacological relevance considering that it is shared by other anti-rheumatic agents (e.g. nonsteroidal antiinflammatory drugs). Thus, we studied the effect of oral administration of FO and corn oil (CO), a vegetable oil with a different composition, on the stability of rat serum proteins, evaluated buy a classical in vitro method based on heat-induced protein denaturation. FO, and, to a lower extent, CO inhibited heat-induced denaturation of rat serum (RS): based on the inhibitory activity (EC50) of the major fatty acids against heat-induced denaturation of RS in vitro, it was possible to speculate the in vivo effects of palmitic acid (C16:0) and eicosapentaenoic acid (EPA, C20:5, n-3) may be more relevant than that of linolenic acid (C18:2). To better investigate this phenomenon, we extracted albumin from the serum of animals treated or not with FO with a one-step affinity chromatography technique, obtaining high purity rat serum albumin preparations (RSA-CTRL and RSA-FO), as judged by SDS-PAGE with Coomassie blue staining. When these RSA preparations were heated at $70^{\circ}C$ for 30 min, it was noted that RSA-FO was much more stable than RSA-CTRL, presumably due to higher number of long chain fatty acids (FA) such as palmitic acid or EPA. In conclusion, we provided evidences that oral administration of FO in the rat stabilizes serum albumin, due to an increase in the number of protein bound long chain fatty acids (e.g. palitic acid and EPA). We speculate that the stabilization of serum albumin and perhaps other proteins could prevent changes of antigenicity due to protein denaturation and glycosylation, which may trigger pathological autoimmune responses, suggesting that this action may be involved in the mode of action of FO in RA and other chronic inflammatory diseases.

• Journal of Life Science
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• v.23 no.4
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• pp.586-594
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• 2013

Post-translational O-GlcNAc modification (O-GlcNAcylation) of serine or threonine is a new protein modulation mechanism. In contrast to the classical glycosylation, O-GlcNAcylation occurs in a one-step transfer of O-GlcNAc on both nuclear and cytoplasmic proteins. In contrast to the general consensus that O-GlcNAc is a final modification, a recent paper (J Proteome Res. 2011 10:2725-2733) showed the presence of O-GlcNAc-P on a synaptic assembly protein AP180. This finding raises a fundamental question about its prevalence. To address this question, we used proteomics to identify those proteins that were phospho-signal enriched by GlcNAc kinase (NAGK). Comparison of pDsRed2-$NAGK_{WT}$-transfected HEK293T cell extract with pDsRed2-$NAGK_{D107A}$-transfected control culture revealed 15 phospho-signal increased spots. Excluding those spots that had no detectable amount of protein expression yielded 7 spots, which were selected for ID determination. Among these, two duplicate spots (two $HSP90{\beta}$ and two ENO1 spots) were shown to be O-GlcNAcylated, two (dUTP nucleotidohydrolase mitochondrial isoform 2, glutathione S-transferase P) were not known to be involved in O-GlcNAcylation, and one (heat shock protein gp96 precursor or grp94) was a glycoprotein. The increase in the phospho-levels of O-GlcNAc by NAGK strongly indicates that these proteins are phosphorylated on O-GlcNAc. Our present data support the idea that O-GlcNAc is not a terminal modification.

• Journal of Life Science
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• v.20 no.5
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• pp.683-690
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• 2010

The yeast strains of Saccharomyces diastaticus produce one of three isozymes of an extracellular glucoamylase I, II or III, a type of exo-enzyme which can hydrolyse starch to generate glucose molecules from non-reducing ends. These enzymes are encoded by the STA1, STA2 and STA3 genes. Another gene, sporulation-specific glucoamylase (SGA), also exists in the genus Saccharomyces which is very homologous to the STA genes. The SGA has been known to be produced in the cytosol during sporulation. However, we hypothesized that the SGA is capable of being secreted to the extracellular region because of about 20 hydrophobic amino acid residues at the N-terminus which can function as a signal peptide. We expressed the cloned SGA gene in S. diastaticus YIY345. In order to compare the biochemical properties of the extracellular glucoamylase and the SGA, the SGA was purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 chromatography. The molecular weight of the intact SGA was estimated to be about 130 kDa by gel filtration chromatography with high performance liquid chromatography (HPLC) column. Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed it was composed of two heterogeneous subunits, 63 kDa and 68 kDa. The deglycosylation of the SGA generated a new 59 kDa band on the SDS-PAGE analysis, indicating that two subunits are glycosylated but the extent of glycosylation is different between them. The optimum pH and temperature of the SGA were 5.5 and $45^{\circ}C$, respectively, whereas those for the extracellular glucoamylase were 5.0 and $50^{\circ}C$. The SGA were more sensitive to heat and SDS than the extracellular glucoamylase.

• Korean Journal of Veterinary Service
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• v.25 no.3
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• pp.245-257
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• 2002

The gene encoding the HN protein from the CBP-1 strain, a heat stable Newcastle disease virus (NDV) isolated from diseased pheasants in Korea, was characterized by reverse transcriptase- polymerase chain reaction(RT-PCR) and the nucleotide and amino acid sequences were analyzed following cloning of the HN gene. In all of the NDV strains studied, a 1.75 kb size cDNA fragment for the HN gene was generated by RT-PCR and smaller specific band sizes harboring the internal portions of the HN gene were also detected by using four pairs of primers. The RT-PCR was sensitive enough to detect viral transcripts when the virus titer was above 25 hemagglutination units. The amplified 1.75 kb cDNA was cloned into a BamHI site of the pVL1393 Baculo transfer vector. The nucleotide sequences of the 1,758 bp HN gene from the CBP-1 strain were determined by the dye terminator cyclic sequencing method. The gene sequences were compared among the strains of CBP-1, Texas GB, Beaudette C, LaSota, B1 and Ulster. The homology of the CBP-1 HN gene to other HN variants was 97.8% to Texas GB, 98.4% to Beaudette C, 95.4% to LaSota, 95.6% to B1 and 90.2% to Ulster. As the deduced 577 amino acid sequences were compared among the strains, the homology for CBP-1 HN appeared to be 96.7% to Texas GB, 97.9% to Beaudette C, 95.5% to LaSota, 95.5% to B1 and 92.7% to Ulster. It was evident that the amino acid sequences included 5 sites for N-asparagine linked glycosylation and 12 cysteine residues. The three conserved leucine residues within the predicted transmembrane domain of the HN protein are amino acid 30, 37 and 44. The three antigenic sites on the HN protein of NDV are amino acids 347(Glu), 481(Asn) and 495(Glu). These data indicate that the genotype of the CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than it is for the LaSota, B1 and Ulster strains.

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.89-100
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• 2002

The nonstructural glycoprotein NSP4, encoded by the 10th gene of rotavirus, has been known to play important roles in viral assembly and pathogenesis. The NSP4 genes of human rotavirus Korean isolates, designated as CBNU/HR-1, CBNU/HR-2, CBNU/HR-3, and CBNU/HR-4, were cloned, sequenced and characterized. Also, the NSP4 gene of the CBNU/HR-1 was expressed in a baculovirus-insect cell system. The sequence data indicated that the NSP4 genes of human rotavirus Korean isolates were 750 or 751 bases in length and encoded one open reading frame of 175 amino acids. Two glycosylation sites were recognized in the NSP4 gene of human rotavirus isolates tested. The NSP4 of CBNU/HR-1, CBNU/HR-3, and CBNU/HR-4 exhibited a high degree of amino acid sequence homology with that of NSP4 genotype B viruses, but a low degree of amino acid sequence homology with that of NSP4 genotype A viruses. However, the NSP4 of CBNU/HR-2 exhibited a high degree of amino acid sequence homology with that of NSP4 genotype A viruses, but a low degree of amino acid sequence homology with that of NSP4 genotype B viruses. The Sf9 cells infected with recombinant baculovirus, inserted with NSP4 gene of CBNU/HR-1, produced specific cytopathic effects and the expressed NSP4 was detected by immunofluorescence staining using NSP4-specific monoclonal antibody(MAb). The expressed NSP4 migrated at 16-26 kDa on SDS-PAGE and reacted with NSP4-specific MAb by Western blotting.

• Molecules and Cells
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• v.44 no.10
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• pp.770-779
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• 2021

Transgenic Arabidopsis thaliana expressing an anti-rabies monoclonal antibody (mAb), SO57, was obtained using Agrobacterium-mediated floral dip transformation. The endoplasmic reticulum (ER) retention signal Lys-Asp-Glu-Leu (KDEL) was tagged to the C-terminus of the anti-rabies mAb heavy chain to localize the mAb to the ER and enhance its accumulation. When the inaccurately folded proteins accumulated in the ER exceed its storage capacity, it results in stress that can affect plant development and growth. We generated T₁ transformants and obtained homozygous T₃ seeds from transgenic Arabidopsis to investigate the effect of KDEL on plant growth. The germination rate did not significantly differ between plants expressing mAb SO57 without KDEL (SO plant) and mAb SO57 with KDEL (SOK plant). The primary roots of SOK agar media grown plants were slightly shorter than those of SO plants. Transcriptomic analysis showed that expression of all 11 ER stress-related genes were not significantly changed in SOK plants relative to SO plants. SOK plants showed approximately three-fold higher mAb expression levels than those of SO plants. Consequently, the purified mAb amount per unit of SOK plant biomass was approximately three times higher than that of SO plants. A neutralization assay revealed that both plants exhibited efficient rapid fluorescent focus inhibition test values against the rabies virus relative to commercially available human rabies immunoglobulins. KDEL did not upregulate ER stress-related genes; therefore, the enhanced production of the mAb did not affect plant growth. Thus, KDEL fusion is recommended for enhancing mAb production in plant systems.