• The Korean Journal of Food And Nutrition
• /
• v.15 no.2
• /
• pp.104-111
• /
• 2002

The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.

• Preventive Nutrition and Food Science
• /
• v.2 no.4
• /
• pp.333-337
• /
• 1997

Trans-cinnamic acid-4-hydroxylase(tC4H) is the first cytochrome P450-dependent monooxygenase of the phenylpropanoid pathway. The roots of avocado seedlings were wounded and examined to determine whether the tC4H would be activated in response to wounding and/or whether tC4H activity be modulated by the application of exogenous p-coumarate. At the specified length of times, the wounded and treated roots were either frozen in liquid nitrogen or used immediately to extract microsomal proteins. The microsomal proteins were subjected to immunoblot analysis using polyclonal antibodies against CYP73 of tC4H gene. In this study, tC4H was induced in wounded roots sealed in bags within 6 hours, and in low level({TEX}$10^{-8}${/TEX}M) of p-coumarate solution within 24 hours, whereas the olution without p-coumarate and high levels of p-coumarate solution repressed tC4H induction in wounded roots. These results indicate that tC4H is induced by wounding in the root of avocado, and is inhibited by the application of exogenous p-coumarate.

• Molecules and Cells
• /
• v.45 no.5
• /
• pp.306-316
• /
• 2022

Chronic stress contributes to the risk of developing depression; the habenula, a nucleus in epithalamus, is associated with many neuropsychiatric disorders. Using genome-wide gene expression analysis, we analyzed the transcriptome of the habenula in rats exposed to chronic restraint stress for 14 days. We identified 379 differentially expressed genes (DEGs) that were affected by chronic stress. These genes were enriched in neuroactive ligand-receptor interaction, the cAMP (cyclic adenosine monophosphate) signaling pathway, circadian entrainment, and synaptic signaling from the Kyoto Encyclopedia of Genes and Genomes pathway analysis and responded to corticosteroids, positive regulation of lipid transport, anterograde trans-synaptic signaling, and chemical synapse transmission from the Gene Ontology analysis. Based on protein-protein interaction network analysis of the DEGs, we identified neuroactive ligand-receptor interactions, circadian entrainment, and cholinergic synapse-related subclusters. Additionally, cell type and habenular regional expression of DEGs, evaluated using a recently published single-cell RNA sequencing study (GSE137478), strongly suggest that DEGs related to neuroactive ligand-receptor interaction and trans-synaptic signaling are highly enriched in medial habenular neurons. Taken together, our findings provide a valuable set of molecular targets that may play important roles in mediating the habenular response to stress and the onset of chronic stress-induced depressive behaviors.

• BMB Reports
• /
• v.45 no.3
• /
• pp.133-140
• /
• 2012

Cancers claim millions of lives each year. Early detection that can enable a higher chance of cure is of paramount importance to cancer patients. However, diagnostic tools for many forms of tumors have been lacking. Over the last few years, studies of chimeric RNAs as biomarkers have emerged. Numerous reports using bioinformatics and screening methodologies have described more than 30,000 expressed sequence tags (EST) or cDNA sequences as putative chimeric RNAs. While cancer cells have been well known to contain fusion genes derived from chromosomal translocations, rearrangements or deletions, recent studies suggest that trans-splicing in cells may be another source of chimeric RNA production. Unlike cis-splicing, trans-splicing takes place between two pre-mRNA molecules, which are in most cases derived from two different genes, generating a chimeric non-co-linear RNA. It is possible that trans-splicing occurs in normal cells at high frequencies but the resulting chimeric RNAs exist only at low levels. However the levels of certain RNA chimeras may be elevated in cancers, leading to the formation of fusion genes. In light of the fact that chimeric RNAs have been shown to be overrepresented in various tumors, studies of the mechanisms that produce chimeric RNAs and identification of signature RNA chimeras as biomarkers present an opportunity for the development of diagnoses for early tumor detection.

• BMB Reports
• /
• v.36 no.6
• /
• pp.538-544
• /
• 2003

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.

• Korean Journal of Breeding Science
• /
• v.41 no.4
• /
• pp.385-390
• /
• 2009

A resveratrol synthase (RS) gene was isolated from peanut (Arachis hypogaea, L. cv. Jinpoong) plants. This gene was placed under the control of the cauliflower mosaic virus 35S promoter (CaMV35S) and introduced into two Korean varieties of potato (Solanum tuberosum L. cvs. Jasim and Jowon) plants by Agrobacterium-mediated gene transfer. Putative transformants were screened by PCR with primers designed from CaMV 35S promoter, NOS terminator and RS gene. Most of selected transgenic potato plants showed the amplification of expected fragments by PCR of genomic DNA with gene-specific primers, while they were absent in untransformed control plants. Expression of the resveratrol synthase gene was also examined by northern blot analysis. The transformants showed a band which was lacking in the control plant, confirming that the introduced gene is transcribed into mRNA in the transformants. The strength of the band, which reflected the level of mRNA expression, differed among the individual transformants. Among the transformants obtained, the highest trans-resveratrol content in the transgenic young leaves of purple-fleshed "Jashim" was $2.11{\mu}gg^{-1}$ fresh weight and that in the microtubers in vitro of purple fleshed "Jashim" was $8.31{\mu}gg^{-1}$ fresh weight. This amount of resveratrol may have a positive biological effect on human health.

• The KIPS Transactions:PartD
• /
• v.18D no.2
• /
• pp.89-100
• /
• 2011

The CNV (Copy Number Variation) which is one of the genetic structural variations in human genome is closely related with the function of gene. In particular, the genome-wide association studies for genetic diseased persons have been researched. However, there have been few studies which infer the genetic function of CNV with normal human. In this paper, we propose the analysis method to reveal the functional relationship between common CNV and genes without considering their genomic loci. To achieve that, we propose the data integration method for heterogeneity biological data and novel measurement which can calculate the correlation between common CNV and genes. To verify the significance of proposed method, we has experimented several verification tests with GO database. The result showed that the novel measurement had enough significance compared with random test and the proposed method could systematically produce the candidates of genetic function which have strong correlation with common CNV.

• BMB Reports
• /
• v.53 no.11
• /
• pp.565-575
• /
• 2020

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades. Among the viral vectors available, gutless adenovirus (GLAd) has been recognized as one of the most promising vectors for in vivo gene delivery. GLAd is constructed by deleting all the viral genes from an adenovirus. Owing to this structural feature, the production of GLAd requires a helper that supplies viral proteins in trans. Conventionally, the helper is an adenovirus. Although the helper adenovirus efficiently provides helper functions, it remains as an unavoidable contaminant and also generates replication-competent adenovirus (RCA) during the production of GLAd. These two undesirable contaminants have raised safety concerns and hindered the clinical applications of GLAd. Recently, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid instead of a helper adenovirus. Utilization of this helper plasmid eliminated the helper adenovirus and RCA contamination in the production of GLAd. HF-GLAd, devoid of helper adenovirus and RCA contaminants, will facilitate its clinical applications. In this review, we discuss the characteristics of adenoviruses, the evolution and production of adenoviral vectors, and the unique features of HF-GLAd as a new platform for gene therapy. Furthermore, we highlight the potential applications of HF-GLAd as a gene delivery vector for the treatment of various inherited genetic diseases.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.49 no.5
• /
• pp.434-439
• /
• 2004

Medicago truncatula is a model plant for molecular genetic studies of legumes and plant-microbe interactions. To accelerate finding of genes that play roles in the early stages of nodulation and stress responses, a trans-genic plant was developed that contains a promoter­reporter fusion. The promoter of rip], a Rhizobium-induced peroxidase gene, was fused to the coding region of $\beta-glucuronidase (GUS)$ gene and inserted into a modified plant transformation vector, pSLJ525YN, in which the bar gene was preserved from the original plasmid but the neomycin phosphotransferase gene was replaced by a polylinker. Transformation of M. truncatula was carried out by vacuum infiltration of young seedlings with Agrobacterium. Despite low survival rates of infiltrated seedlings, three independent transformants were obtained from repeated experiments. Southern blot analyses revealed that 7 of 8 transgenic plants of the T 1 generation contained the bar gene whereas 6 $T_1$ plants contained the GUS gene. These results indicate that vacuum infiltration is an effective method for transformation of M. truncatula. The progeny seeds of the transgenic plants will be useful for mutagenesis and identification of genes that are placed upstream and may influence the expression of rip] in cellular signaling processes including nodulation.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.2
• /
• pp.237-245
• /
• 2013

Canthaxanthin (cx) is a potent antioxidant that is chemically synthesized at the industrial scale and has imperative applications in the cosmetic and feed industries. An orange pigmented mesophilic bacterium, designated as K44, was isolated from soil samples of Kargil, India. Biochemical tests, 16S rRNA gene sequencing, and FAME analysis of the bacterium indicated it to belong in the genus Dietzia and is distinct from human isolates. The strain showed 98% 16S rRNA gene sequence homology with Dietzia maris DSM 43102. High-performance liquid chromatography profile of the pigments isolated from K44 showed two major peaks absorbing at 465.3 and 475 nm. The liquid chromatography-mass spectrometry (LC-MS) analysis of both these peaks revealed their m/z to be 564. The molecular weights, LC-MS/MS fragmentation patterns, and ${\lambda}_{max}$ of these fractions corresponded to all-trans- (475 nm) and 9-cis-(465.3 nm) cx isomers. The antioxidant activities of cis- and trans-cx isomers isolated from this bacterium were found to differ, where the cis-isomer showed higher free radical, superoxide radical, and reactive oxygen species scavenging activities than the alltrans- isomer, suggesting that 9-cis-cx is more effective as an antioxidant than the all-trans-cx.