• Korean Journal of Microbiology
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• v.55 no.2
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• pp.103-111
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• 2019

DNA methylation is involved in diverse processes in bacteria, including maintenance of genome integrity and regulation of gene expression. CcrM, the DNA methyltransferase conserved in Alphaproteobacterial species, carries out $N^6$-adenine or $N^4$-cytosine methyltransferase activities using S-adenosyl methionine as a co-substrate. Celeribacter marinus IMCC12053 from the Alphaproteobacterial group was isolated from a marine environment. Single molecule real-time sequencing method (SMRT) was used to detect the methylation patterns of C. marinus IMCC12053. Gibbs motif sampler program was used to observe the conversion of adenosine of 5'-GANTC-3' to $N^6$-methyladenosine and conversion of $N^4$-cytosine of 5'-GpC-3' to $N^4$-methylcytosine. Exocyclic DNA methyltransferase from the genome of strain IMCC12053 was chosen using phylogenetic analysis and $N^4$-cytosine methyltransferase was cloned. IPTG inducer was used to confirm the methylation activity of DNA methylase, and cloned into a pQE30 vector using dam-/dcm- E. coli as the expression host. The genomic DNA and the plasmid carrying methylase-encoding sequences were extracted and cleaved with restriction enzymes that were sensitive to methylation, to confirm the methylation activity. These methylases protected the restriction enzyme site once IPTG-induced methylases methylated the chromosome and plasmid, harboring the DNA methylase. In this study, cloned exocyclic DNA methylases were investigated for potential use as a novel type of GpC methylase for molecular biology and epigenetics.

• Journal of Animal Science and Technology
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• v.62 no.5
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• pp.614-627
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• 2020

During the course of this trial, our team assessed the influence of protease upon the growth performance, the nutrient digestibility, and the expression of growth-related genes and amino acid transporters within the liver, muscle, and small intestines of broilers. During the first step, our team allocated 600 broilers into four dietary treatments for a period of 35 days in order to measure the growth performance and nutrient digestibility of the broilers selected. The separate treatments contained 10 replicates (15 birds per replicate). The treatments were composed of: 1) CON, basal diet; 2) T1, basal diet + 0.03% protease; 3) T2, basal diet + 0.06% protease; and 4) T3, basal diet + 0.09% protease. Next, the broiler chick sample tissue was harvested from the CON and T3 groups in order to conduct gene expression analysis following the feeding trials the broilers underwent. Our team discovered that the broilers fed protease diets possessed increased body weight and an average daily gain, but conversely, had lower feed conversion ratios when their dietary protease levels increased from 0% to 0.09% (p < 0.05). Additionally, significant linear improvements were identified among the nutrient digestibility of dry matter, crude protein, energy, and amino acids within broilers supplied with protease diets when contrasted and compared with broilers supplied with the basal diet (p < 0.05). In addition, the gene expression of the genes IGF1, IGF2, GH, and LEP in the liver, and the genes MYOD1 and MYOG in the breast muscles, was significantly increased after broilers were fed with a protease diet as compared to broilers that subsisted on a basal diet (p < 0.05). Protease supplementation also raised the expression levels within these amino acid transporters: SCL6A19, SLC7A1, SLC7A7, SLC7A2, SLC7A6, SLC7A9, and SLC15A1, located in the small intestine, when compared to the basal diet (p < 0.05). Our results suggest that protease supplementation in their diet improved the growth performance of broilers via an increase in the expression growth-related genes within broiler liver and muscle tissue. In addition, protease supplementation enhanced broiler digestibility via the upregulation of amino acid transporter expression within the small intestine.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.37 no.3
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• pp.288-298
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• 1992

Since the major important factors limiting plant growth and crop productivity are environmental stresses, of which low temperature is the most serious. It has been well known that many physiological processes are alterant in response to the environmental stress. With regard to the relationship between plant hormones and the regulation of chilling tolerance in rice seedlings, the major physiological roles of plant hormones: abscisic acid, ethylene and polyamines are evaluated and discussed in this paper. Rice seedlings were grown in culture solution to examine the effect of such plant hormones on physiological characters related to chilling tolerance and also to compare the different responses among tested cultivars. Intact seedlings about 14 day-old were chilled at conditions of 5$^{\circ}C$ and 80% relative humidity for various period. Cis-(＋)-ABA content was measured by the indirect ELISA technique. Polyamine content and ethylene production in leaves were determined by means of HPLC and GC respectively. Chilling damage of seedlings was evaluated by electrolyte leakage, TTC viability assay or servival test. Our experiment results described here demonstrated the physiological functions of ABA, ethylene, and polyamines related to the regulation of chilling tolerance in rice seedlings. Levels of cis-(＋)-ABA in leaves or xylem sap of rice seedlings increased rapidly in response to 5$^{\circ}C$ treatment. The tolerant cultivars had significant higher level of endogenous ABA than the sensitive ones. The ($\pm$)-ABA pretreatment for 48 h increased the chilling tolerance of the sensitive indica cultivar. One possible function of abscisic acid is the adjustment of plants to avoid chilling-induced water stress. Accumulation of proline and other compatible solutes is assumed to be another factor in the prevention of chilling injuies by abscisic acid. In addition, the expression of ABA-responsive gene is reported in some plants and may be involving in the acclimation to low temperature. Ethylene and its immediate precusor, 1-amincyclopropane-1-carboxylic acid(ACC) increased significantly after 5$^{\circ}C$ treatment. The activity of ACC synthase which converts S-adenosylmethionine (SAM) to ACC enhanced earlier than the increase of ethylene and ACC. Low temperature increased ACC synthase activity, whereas prolonged chilling treatment damaged the conversion of ACC to ethylene. It was shown that application of Ethphon was beneficial to recovering from chilling injury in rice seedlings. However, the physiological functions of chilling-induced ethylene are still unclear. Polyamines are thought to be a potential plant hormone and may be involving in the regulation of chilling response. Results indicated that chilling treatment induced a remarkable increase of polyamines, especially putrescine content in rice seedlings. The relative higher putrescine content was found in chilling-tolerant cultivar and the maximal level of enhanced putrescine in shoot of chilling cultivar(TNG. 67) was about 8 folds of controls at two days after chilling. The accumulation of polyamines may protect membrane structure or buffer ionic imbalance from chilling damage. Stress physiology is a rapidly expanding field. Plant growth regulators that improve tolerance to low temperature may affect stress protein production. The molecular or gene approaches will help us to elucidate the functions of plant hormones related to the regulation of chilling tolerance in plants in the near future.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

• Journal of Plant Biotechnology
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• v.1 no.1
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• pp.20-30
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• 1999

In order to modify lipid production of Perilla qualitatively as well as quantitatively by genetic engineering, genes involved in carbon metabolism were isolated and characterized. These include acyl-ACP thioesterases from Perilla frutescens and Iris sp., four different $\beta$-ketoacyl- ACP synthases from Perilla frutescens, and two $\Delta$15 a-cyl-ACP desaturases(Pffad7, pffad3). Δ15 acyl-ACP desa turase (Δ15-DES) is responsible for the conversion of linoleic acid (18:2) to $\alpha$-linolenic acid (ALA, 18:3). pffad 3 encodes Δ15 acyl-desaturase which is localized in ER membrane. On the other hand, Pffad7 encodes a 50 kD plastid protein (438 residues), which showed highest sequence similarity to Sesamum indicum fad7 protein. Northern blot analysis revealed that the Pffad7 is highly expressed in leaves but not in roots and seeds. And Pffad3 is expressed throughout the seed developmental stage except very early and fully mature stage. We constructed Pffad7 gene under 355 promoter and Pffad3 gene under seed specific vicillin promoter. Using Pffad7 construct, Perilla, an oil seed crop in Korea, was transformed by Agrobacterium leaf disc method. $\alpha$-linolenic acid contents increased in leaves but decreased in seeds of transgenic Perilla. Currently, we are transforming Perilla with Pffad3 construct to change Perilla seed oil composition. We isolated three ADP-glucose pyrophosphorylase (AGP) genes from Perilla immature seed specific cDNA library. Nucleotide sequence analysis showed that two of three AGP (Psagpl, Psagp2) genes encode AGP small subunit polypeptides and the remaining (Plagp) encodes an AGP large subunit. PSAGPs, AGP small subunit peptide, form active heterotetramers with potato AGP large subunit in E. coli expressing plant AGP genes.

• Journal of Life Science
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• v.24 no.12
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• pp.1364-1370
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• 2014

Ascorbic acid (AA) is a multifunctional metabolite in plants that is essential for plant development and growth. We examined the effect of AA, an antioxidant, on the gravitropic response of primary roots in maize. The application of $10^{-3}$ M AA to the elongation zone did not affect the gravitropic response and slightly inhibited the root growth. However, treatment with both $10^{-5}$ M and $10^{-3}$ M AA at the root tip increased the gravitropic response and inhibited root growth. Differences in indole-3- acetic acid (IAA) activity between the upper and lower hemispheres of the root resulted in differential elongation along the horizontal root. Roots are extremely sensitive to IAA, and increasing the amount of IAA in the lower hemisphere of the root inhibited elongation. Therefore, we examined the effect of IAA in the presence of AA. The inhibitory effect of AA on the gravitropic response was greater in combination with IAA. To understand the role of AA in the regulation of root growth and the gravitropic response, we measured ethylene production in the presence of AA in the primary roots of maize. AA stimulated ethylene production via the activation of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene, which regulates the conversion of ACC to ethylene. These results suggest that AA alters the gravitropic response of maize roots through modification of the action of ethylene.

• KSBB Journal
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• v.20 no.2 s.91
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• pp.93-99
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• 2005

Polyunsaturated fatty acids, that is PUFAs, are important constituents of membranes particularly found in the retina and central nervous system. In microorganism-based PUFAs biosynthesis, the genus Thraustochytrids is well evaluated for their potential as a promising candidate in the practical production of PUFAs, such as AA and DHA. In this study, we attempted to optimize a method of total nucleic acid extraction from this microorganism as a preliminary experiment. Using the extracted nucleic acid and degenerated primers for direct PCR, we isolated a $\Delta5$ desaturase gene that contained 1320-nucleotide and encoded 439 amino acids. This gene exhibited an expected function, when expressed in P. pastoris in the presence of appropriate exogenous substrate, as an evidence for $\Delta5$ desaturase activity (conversion of DGLA to AA). These results and information could provide a basis for the construction of engineered strains suitable for the practical production of PUFAs.

• Journal of Life Science
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• v.31 no.5
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• pp.520-526
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• 2021

Microbial protein is one of the sources of protein in the rumen and can also be the source of glutamate production. Glutamic acid is used as fuel in the metabolic reaction in the body and the synthesis of all proteins for muscle and other cell components, and it is essential for proper immune function. Moreover, it is used as a surfactant, buffer, chelating agent, flavor enhancer, and culture medium, as well as in agriculture for such things as growth supplements. Glutamic acid is a substrate in the bioproduction of gamma-aminobutyric acid (GABA). This review provides insights into the role of glutamic acid and glutamic acid-producing microorganisms that contain the glutamate decarboxylase gene. These glutamic acid-producing microorganisms could be used in producing GABA, which has been known to regulate body temperature, increase DM intake and milk production, and improve milk composition. Most of these glutamic acid and GABA-producing microorganisms are lactic acid-producing bacteria (LAB), such as the Lactococcus, Lactobacillus, Enterococcus, and Streptococcus species. Through GABA synthesis, succinate can be produced. With the help of succinate dehydrogenase, propionate, and other metabolites can be produced from succinate. Furthermore, clostridia, such as Clostridium tetanomorphum and anaerobic micrococci, ferment glutamate and form acetate and butyrate during fermentation. Propionate and other metabolites can provide energy through conversion to blood glucose in the liver that is needed for the mammary system to produce lactose and live weight gain. Hence, health status and growth rates in ruminants can be improved through the use of these glutamic acid and/or GABA-producing microorganisms.

• Animal Bioscience
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• v.35 no.1
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• pp.64-74
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• 2022

Objective: This study was conducted to investigate the effects of dietary multi-strain probiotic (MSP) (Bacillus coagulans Unique IS2 + Bacillus subtillis UBBS14 + Saccharomyces boulardii Unique 28) on performance, gut morphology and expression of nutrient transporter related genes in broiler chickens. Methods: A total of 256 (4×8×8) day-old CARIBRO Vishal commercial broiler chicks of uniform body weight were randomly distributed into four treatments with 8 replicates each and having eight chicks in each replicate. Four dietary treatments were T₁ (negative control-basal diet), T₂ (positive control-antibiotic bacitracin methylene disalicylate at 20 mg/kg diet), T₃ (MSP at 10⁷ colony-forming unit [CFU]/g feed), and T₄ (MSP at 10⁸ CFU/g feed). Results: During 3 to 6 weeks and 0 to 6 weeks, the body weight gain increased significantly (p<0.05) in T₃ and T₄ groups. The feed intake significantly (p<0.05) reduced from T₁ to T₃ during 0 to 3 weeks and the feed conversion ratio also significantly (p<0.05) improved in T₃ and T₄ during 0 to 6 weeks. The humoral and cell mediated immune response and the weight of immune organs were also significantly (p<0.05) improved in T₃ and T₄. However, significant (p<0.05) dietary effects were observed on intestinal histo-morphometry of ileum in T₃ followed by T₄ and T₂. At 14 d post hatch, the relative gene expression of glucose transporter (GLUT5), sodium-dependent glucose transporter (SGLT₁) and peptide transporter (PepT₁) showed a significant (p<0.05) up-regulating pattern in T₂, T₃, and T₄. Whereas, at 21 d post hatch, the gene expression of SGLT₁ and PepT₁ was significantly (p<0.05) downregulated in MSP supplemented treatments T₃ and T₄. Conclusion: The supplementation of MSP at 10⁷ CFU/g diet showed significant effects with improved performance, immune response, gut morphology and expression of nutrient transporter genes. Thus, the MSP could be a suitable alternative to antibiotic growth promoters in chicken diets.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.21 no.1
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• pp.22-27
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• 2021

Propionic acidemia (PA) is an inherited autosomal recessive disorder, due to the deficiency of propionyl-CoA carboxylase (PCC). PCC is the enzyme which catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA, and it is critical for the metabolism of amino acids, odd-chain fatty acids, and side chains of cholesterol. The clinical manifestations present mostly at the neonatal period with life-threatening metabolic acidosis and hyperammonemia. Here, we described a case of a 16-year-old Korean boy with late-onset PA who presented with embolic cerebral infarction due to dilated cardiomyopathy (DCMP) with left ventricular noncompaction. And he has family history of sudden cardiac death, so we performed metabolic screening and genetic tests. Elevated levels of 3-hydroxypropionic acid, methylcitric acid and propionylglycerine were detected in urine. Plasma acylcarnitine profile showed elevated propionylcarnitine (C3). Diagnosis of PA was confirmed by genetic analysis, which revealed compound heterozygous mutations, c.[1151T>G] (p.[Phe384Cys]) and c.[1228C>T] (p.[Arg410Trp]) in PCCB gene. His heart function is in improving state and the results of biochemical analysis are stable with heart failure medication and metabolic managements. We present a case of patient without episodes of metabolic decompensation who manifests DCMP as the first symptom of PA.