• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.65-74
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• 2021

Serine proteases are the most versatile proteolytic enzymes with tremendous applications in various industrial processes. This study was designed to investigate the biochemical properties, critical residues, and the catalytic potential of alkaline serine protease using in-silico approaches. The primary sequence was analyzed using ProtParam, SignalP, and Phyre2 tools to investigate biochemical properties, signal peptide, and secondary structure, respectively. The three-dimensional structure of the enzyme was modeled using the MODELLER program present in Discovery Studio followed by Molecular Dynamics simulation using GROMACS 5.0.7 package with CHARMM36m force field. The proteolytic potential was measured by performing docking with casein- and keratin-enriched residues, while the effect of the inhibitor was studied using phenylmethylsulfonyl fluoride, (PMSF) applying GOLDv5.2.2. Molecular weight, instability index, aliphatic index, and isoelectric point for serine protease were 39.53 kDa, 27.79, 82.20 and 8.91, respectively. The best model was selected based on the lowest MOLPDF score (1382.82) and DOPE score (-29984.07). The analysis using ProSA-web revealed a Z-score of -9.7, whereas 88.86% of the residues occupied the most favored region in the Ramachandran plot. Ser327, Asp138, Asn261, and Thr326 were found as critical residues involved in ligand binding and execution of biocatalysis. Our findings suggest that bioengineering of these critical residues may enhance the catalytic potential of this enzyme.

• Mycobiology
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• v.49 no.6
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• pp.582-588
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• 2021

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.

• Investigative Magnetic Resonance Imaging
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• v.1 no.1
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• pp.135-141
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• 1997

Purpose: To assess the usefulness of breath-hold fast MR imaging of liver with fat suppression (FS) by application of chemical saturation technique in the diagnosis of regional fatty changes suspected in sonography. Materials and Methods: Thirteen patients who had focal lesions with diffuse, homogeneous signal changes after FS through chemical saturation technique without additional changes of imaging parameter during MR imaging of liver were selected. T1-weighted fast low-angle shot and T2-weighted turbo spin-echo sequences were obtained with or without FS during each single breath-holding session. Subjective changes of signal intensity between the pre-FS and the FS images were compared with the sonographic findings in each lesion. Results: Seven lesions of decreased signal intensity after FS on T1 or T2-weighted images, including three lesions only at FS T1 images, were regarded as focal fat infiltration. All seven lesions had compatible sonographic findings as homogenously echogenic areas. Another six lesions of subjectively increased signal intensity including two lesions only at FS T2 images were regarded as focal fat sparing. All six lesions had sonographic findings as homogenous echo poor areas suggesting focal fat sparing. In cases regarded as fat infiltration, score changes were more prominent at FS T1 images than FS T2 images(p=0.0002). In cases regarded as fat sparing, score changes were more prominent at FS T2 images than FS T1 images(p=0.042). Conclusion: Breath-hold fast T1 and T2-weighted MR imaging with and without chemical saturation pre-pulse may be sufficient for characterization of regional fatty changes in the differential diagnosis of focal hepatic lesion found at sonography.

• Journal of fish pathology
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• v.6 no.2
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• pp.93-101
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• 1993

The RNAI mutation of Saccharomyces cerevisia is a recessive and temperature sensitive lethal mutation which interferes with the production of mRNA, rRNA, and tRNA. However, the precise role of RNAI gene have not been revealed until yet. We have cloned rna1-1 mutant gene from rna1-1 mutant yeast strain(R49 ; trpl, ura3-52, rna1-1). The 3.4kb BglII fragment of wild type RNAI clone(81-2-6) contains whole RNAI gene. The genomic southern blotting with BglII digested R49 genomic DNA as a probe shows the unique and identical band with wild type 3.4kb BglII fragment. Therefore, We prepared partial BglII genomic library(3~4kb BglII fragments) into BamH I site of pUC19. The rna 1-1 mutant clone was screened with Digoxigenin(DIG)-lableled probe by high density colony hybridization. The 5'-flanking region of rna1-1 gene was sequenced by dideoxy chain termination method. The 5'-flanking sequence of RNAI gene contains three TATA-like sequence ; TAATA, TATA and TTTTAA at position of -67, -45, and -36 from first ATG codon respectively. The 5'-flanking region of wild type RNA I gene from ATG codon to -103nt was deleted with Bal31 exonuclease digestion, generating $pUC{\Delta}$/RNA I. After constructing $pYEP{\Delta}RNA$ I (consists of -103nt deleting RNA I gene, URA3 gene, $2{\mu}m$ rep. origin), pYEPrna1-1(consists of Xba I fragment of pUCrna1-1. URA3 gene, $2{\mu}m$ rep. origin), and pYEPRNAI. each plasmid was transformed into host strain(trpl, ura3-52, rna1-1) by electroporation, respectively. Yeast transformant carrying $pYEP{\Delta}RNA$ I did not complement the thermal sensitivity of rna1-1 gene. It means that TATA-like sequences in 5'-flanking region is not TATA sequence for transcribing RNAI gene and there may be other essential sequence in upstream region for the transcription of RNAI gene.

• Bulletin of the Korean Chemical Society
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• v.34 no.7
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• pp.2117-2124
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• 2013

The binding properties and sequence selectivities of ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ (bip = 4,4'-biphenylene (imidazo [4,4-f][1,10]phenanthroline) complexes with $poly[d(A-T)_2]$ and $poly[d(G-C)_2]$ were investigated using conventional spectroscopic methods. When bound to $poly[d(A-T)_2]$, a large positive circular dichroism (CD) spectrum was induced in absorption region of the bridging moiety for both the ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complexes, which suggested that the bridging moiety sits in the minor groove of the polynucleotide. As luminescence intensity increased, decay times became longer and complexes were well-protected from the negatively charged iodide quencher compared to that in the absence of $poly[d(A-T)_2]$. These luminescence measurements indicated that Ru(II) enantiomers were in a less polar environment compared to that in water and supported by minor groove binding. An angle of $45^{\circ}$ between the molecular plane of the bridging moiety of the ${\Delta}{\Delta}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complex and the local DNA helix axis calculated from reduced linear dichroism ($LD^r$) spectrum further supported the minor groove binding mode. In the case of ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complex, this angle was $55^{\circ}$, suggesting a tilt of DNA stem near the binding site and bridging moiety sit in the minor groove of the $poly[d(A-T)_2]$. In contrast, neither ${\Delta}{\Delta}$-nor ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complex produced significant CD or $LD^r$ signal in the absorption region of the bridging moiety. Luminescence measurements revealed that both the ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complexes were partially accessible to the $I^-$ quencher. Furthermore, decay times became shorter when bis-Ru(II) complexes bound to $poly[d(G-C)_2]$. These observations suggest that both the ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complexes bind at the surface of $poly[d(G-C)_2]$, probably electrostatically to phosphate group. The results indicate that ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ are able to discriminate between AT and GC base pairs.

• Investigative Magnetic Resonance Imaging
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• v.11 no.2
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• pp.87-94
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• 2007

Purpose: The purpose of this study was to evaluate the usefulness of fast inversion recovery (FIR) and magnetization-prepared three dimensional gradient echo sequence (3D GRE) T1-weighted sequences for neonatal brain imaging compared with spin echo (SE) sequence in a 3T MR unit. Materials and Methods: T1-weighted axial SE, FIR and 3D GRE sequences were evaluated from 3T brain MR imaging in 20 neonates. The signal-to-noise ratio (SNR) of different tissues was measured and contrast-to-noise ratios (CNR) were determined and compared in each of the sequences. Visual analysis was carried out by grading gray-white matter differentiation, myelination, and artifacts. The Wilcoxon signed ranked test was used for evaluation of the statistical significance of CNR differences between the sequences. Results: Among the three sequences, the 3D GRE had the best SNRs. CNRs obtained with FIR and 3D GRE were statistically superior to those obtained with SE; these CNRs were better on the 3D GRE compared to the FIR. Gray to white matter differentiation and myelination were better delineated on the FIR and 3D GRE than the SE. However, motion artifacts were more commonly observed on the 3D GRE and flow-related artifacts of vessels were frequently seen on the FIR. Conclusion: FIR and 3D GRE are valuable alternative T1-weighted sequences to conventional SE imaging of the neonatal brain at 3T providing superior image quality.

• Investigative Magnetic Resonance Imaging
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• v.1 no.1
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• pp.142-147
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• 1997

Purpose: To compare the effectiveness of the in-phase (IP) sequence and the opposed-phase (Op) sequence in the detection of focal hepatic lesions in the single breath-hold hepatic MR imaging with fast gradient T1-weighted pulse sequences. Materials and Methods: IP and OP T1-weighted breath-hold imaging was performed using fast gradient echo sequences in 45 patients referred for known focal hepatic lesions, in which 78 lesions were detected. Three blind readers independently reviewed the images for lesion detectability. The signal-to-noise ratio (SNR) of the liver, the lesion-to-liver contrast-to-noise ratio (CNR) and the liver-to-spleen CNR were also compared. A consensus was reached by three readers to determine which sequence is better in image quality. Results: On OP images, 61(78%), 61(78%), and 63(89%) lesions were correctly identified for reader 1, 2 and 3, respectively. On IP images, 66(85%), 65(83%), and 65(93%) lesions were detected for each reader, respectively. When two image sets were combined, 71(91 %), 69(88 %), and 76(97%) lesions respectively were detected for each reader. In cases of hepatocellular carcinoma, liver-to-Iesion CNR was greater on the OP images(p (0.05), but in other lesions significant difference was not demonstrated. Liver-to-spleen CNR was higher on OP images(p ( 0.1), but the SNR of the liver was higher on the IP images. Conclusion: Use of both IP and OP imaging can be helpful to avoid erroneous missing of some focal hepatic lesions.

• Journal of Life Science
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• v.30 no.10
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• pp.844-850
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• 2020

Disaster-related extreme weather is rapidly increasing due to climate change. In Korea, typhoons accompanied by rainfall usually approach in August and September, causing great damage. The purpose of this study is to find a gene that regulates the heading date of rice in order to avoid loss of harvest from climate change and typhoons. Cheongcheong/Nagdong doubled haploid (CNDH) was used as the plant material to investigate the location of heading-related genes using QTL and sequence analysis by cloning the gene. In the distribution chart, the heading dates, culm lengths, panicle lengths, numbers of panicles, and 1,000-grain weights all have normal distributions. QTL analysis found 13 contigs on chromosome 8. One QTL, named qHd8, was detected on chromosome 8. The range at qHd8 was approximately 7.7 cM, with RM72 and RM404 markers near the peak. There were 13 contigs and 1 ORF. Protein sequence analysis showed that rice was similar to Os08g0341700, AtSFH13, and AtSFH7 proteins. Os08g0341700, which is involved in signal transduction, is similar to phosphatidylinositol transfer-like protein II, and complete information is not available, but it is believed to play a role in the phosphatidylinositol-specific signaling pathway related to Sec14P.

• Journal of the korean academy of Pediatric Dentistry
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• v.33 no.3
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• pp.401-410
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• 2006

Dental caries results from localized demineralization of tooth enamel by acids of bacterial origin produced from the fermentation of dietary sugars. A group of related oral bacteria, collectively known as mutans streptococci, are implicated as the primary etiological agents of human caries. Within this group, Streptococcus mutans has been known as a causative agent for dental caries. As well as acid production yielding the demineralization of tooth enamel, adherence and colonization of S. mutans to the teeth are also important for their virulence Cell-surface fibrillar proteins, which mediate adherence to the salivary pellicle are virulence components of mutans streptococci, and primary candidates for a human caries vaccine. Here we report that the AgI/II gene from S. mutans GS-5 were cloned by PCR amplification of the bacterial chromosomal DNA and the integrity of cloned genes were confirmed by nucleotide sequencing. Sequence analyses showed the sequence alignment of 280 nucleotides between the cloned AgI/II and the reported sequence of S. mutans GS-5 showed the perfect match The cloned genes which signal nucleotide was truncated, were transferred into bacterial expression vector and the recombinant proteins were purified as His-tag fusion proteins In order to generate polyclonal antibodies against the recombinant proteins, AgI/II mr, some $100{\mu}g$ of the proteins was injected into mice three times. It can be used for an effective vaccine production to prevent dental caries caused by pathogenic S. mutans.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.555-562
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• 2012

PCR was performed to analyze the ${\beta}$-lactamase genes carried by ampicillin-resistant Vibrio spp. strains isolated from marine environments in Korea between 2006 and 2009. All 36 strains tested showed negative results in PCR with the primers designed from the nucleotide sequences of various known ${\beta}$-lactamase genes. This prompted us to screen new ${\beta}$-lactamase genes. A novel ${\beta}$-lactamase gene was cloned from Vibrio alginolyticus KV3 isolated from the aquaculture water of Geoje Island of Korea. The determined nucleotide sequence (VAK-3 ${\beta}$-lactamase) revealed an open reading frame (ORF) of 852 bp, encoding a protein of 283 amino acids (aa), which displayed low homology to any other ${\beta}$-lactamase genes reported in public databases. The deduced 283 aa sequence of VAK-3, consisting of a 19 aa signal peptide and a 264 aa mature protein, contained highly conserved peptide segments specific to class A ${\beta}$-lactamases including the specific amino acid residues STFK (62-65), SDN (122-124), E (158), and RTG (226-228). Results from PCR performed with primers specific to the VAK-3 ${\beta}$-lactamase gene identified 3 of the 36 isolated strains as V. alginolyticus, Vibrio cholerae, and Photobacterium damselae subsp. damselae, indicating the utilization of various ${\beta}$-lactamase genes including unidentified ones in ampicillin-resistant Vibrio spp. strains from the marine environment. In a mating experiment, none of the isolates transfered the VAK-3 ${\beta}$-lactamase gene to the Escherichia coli recipient. This lack of mobility, and the presence of a chromosomal acyl-CoA flanking sequence upstream of the VAK-3 ${\beta}$-lactamase gene, led to the assumption that the location of this new ${\beta}$-lactamase gene was in the chromosome, rather than the mobile plasmid. Antibiotic susceptibility of VAK-3 ${\beta}$-lactamase was indicated by elevated levels of resistance to penicillins, but not to cephalosporins in the wild type and E. coli harboring recombinant plasmid pKV-3, compared with those of the host strain alone. Phylogenetic analysis showed that VAK-3 ${\beta}$-lactamase is a new and separate member of class A ${\beta}$-lactamases.