• KSBB Journal
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• 제19권1호
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• pp.42-49
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• 2004

Racemic epoxide에 대한 입체선택적 가수분해능을 가지고 있는 곰팡이, Aspergillus niger LK로부터 epoxide hydrolase (EH, EC 3.3.2.3) 유전자의 진화적 유연관계 분석을 행하였다. A. niger LK의 EH 염기서열로부터 유추한 EH 단백질 아미노산 서열은 여러 박테리아의 EH들 및 포유동물의 microsomal EH들과 유의적인 유사성을 가지고 있었으며 a/$\beta$ hydrolase fold family에 속하였다. A. niger LK의 EH 단백질의 입체구조예측은 Protein Data Bank에 수록된 lqo7의 3D 결정구조와 90.6% identity를 가지는 것으로 나타났으며 다른 EH들의 아미노산 서열비교를 행한 결과 Asp$^{192}$ , Asp$^{348}$ 및 His$^{374}$ 이 catalytic triad를 구성하고 있는 것으로 추정되었다. 여러 생물종의 EH서열을 기능적 및 구조적 domain 서열을 기초로 하여 multiple sequence alignment를 행하고 Neighbor-Joining/UPGMA method를 이용하여 계통수를 복원한 결과 다른 생물종들의 EH와의 진화거리는 서로 1.841∼2.682로 멀었으나 EH의 기능을 가지기 위한 oxyanion hole 및 a/$\beta$ hydrolase fold family의 catalytic triad는 잘 보존되고 있어 공통조상으로부터 진화되어 왔음을 알 수 있었다.

• BMB Reports
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• 제41권6호
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• pp.448-454
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• 2008

Two full-length cDNAs, PutPMP3-1 and PutPMP3-2, encoding PMP3 family proteins were isolated from Puccinellia tenuiflora, a monocotyledonous halophyte. Expression of both genes was induced by low temperature, salt stress, dehydration, ABA, and $NaHCO_3$. Transcripts of PutPMP3-2 were more strongly induced by these stresses relative to those of PutPMP3-1, particularly under low temperature and dehydration conditions. Expression of PutPMP3-1 and PutPMP3-2 in yeast mutants lacking the PMP3 gene can functionally complement the membrane hyper-polarization and salt sensitivity phenotypes resulting from PMP3 deletion. To compare the functions of PutPMP3-1 and PutPMP3-2, the orthologous genes in rice (OsLti6a and OsLti6b) were isolated. Both OsLti6a and OsLti6b could functionally complement the loss of PMP3 in yeast. PutPMP3-2 and OsLti6a were more effective in reversing membrane hyperpolarization than PutPMP3-1 and OsLti6b. However, the four yeast transformants each showed similar levels of salt tolerance. These results imply that these PMP3 family members don't function identically under different stress tolerance conditions.

• 한국수산과학회지
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• 제48권6호
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• pp.913-919
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• 2015

In vertebrates, the vitamin D receptor (VDR), a member of the nuclear receptor superfamily, binds the biologically active ligand $1{\alpha},25-(OH)_2$-vitamin $D_3$ (1,25 $D_3$). Nearly all vertebrates, including Agnatha, possess a VDR with high ligand selectivity for 1,25 $D_3$ and related metabolites. Although a putative ancestral VDR gene is present in the genome of the chordate invertebrate Ciona intestinalis, the functional characteristics of marine invertebrate VDR are still obscure. To elucidate the ascidian Halocynthia roretzi VDR (HrVDR), we cloned full-length HrVDR cDNA and investigated the transcriptional activity of HrVDR in HEK293 cells. HrVDR consists of 1,680 nucleotides (559 amino acids [aa]), including a short N-terminal region (A/B domain; 26 aa), DNA-binding domain (C domain; 72 aa), hinge region (D domain; 272 aa), and C-terminal ligand-binding domain (E domain; 161 aa). The amino acid sequence identity of HrVDR was greatest to that of C. intestinalis VDR (56%). In the luciferase reporter assays, the transcriptional activity of HrVDR was not significantly increased by 1,25 $D_3$, whereas the farnesoid X receptor agonist GW4064 increased the transactivation of HrVDR. These results suggest the presence of a novel ligand for and a distinct ligand-binding domain in ascidian VDR.

• BMB Reports
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• 제39권6호
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• pp.749-758
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• 2006

The cytosolic members of the HSP70 family of proteins play key roles in the molecular chaperone machinery of the cell. In the study we cloned and sequenced the full-length cDNA of Delia antiqua HSP70 gene, which is 2461 bp long and encodes 643 a.a. with a calculated molecular mass of 70,787 Da. We investigated gene copies of cytosolic HSP70 members of 4 insect species with complete genome available, and found that they are quite variable with species. In order to characterize this protein we carried out an alignment and a phylogenetic analysis with 41 complete protein sequences from insects. The analysis divided the cytosolic members of the family into two classes, HSP70 and HSC70, distinguishable on the basis of 15 residues. HSP70 class members were slightly shorter in length and smaller in molecular mass relative to the HSC70 class members, and the conservative and functional regions in these sequences were documented. Mainly, we investigated the expression of Delia antiqua HSP70 gene, in response to diapauses and thermal stresses. Both summer and winter diapauses elevated HSP70 transcript levels. Cold-stress led to increased HSP70 expression levels in summer- and winter-diapausing pupae, but heat-stress elevated the levels only in the winter-diapausing pupae. In all cases, the expression levels, after being elevated, gradually decreased with time. HSP70 expression was low in non-diapausing pupae but was up-regulated following cold- and heat-stresses. Heat-stress gradually increased the mRNA level with time whereas cold-stress gradually decreased levels after an initial increase.

• 폴리머
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• 제28권2호
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• pp.177-184
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• 2004

고분자-점토 나노복합재는 적은 양의 점토 함유만으로도 물리적, 기계적 특성 등의 물성 증대 효과를 기대할 수 있다 고분자-점토 나노복합재의 일반적인 제조방법으로는 층간 삽입법과 직접 중합법으로 나눌 수 있다. 본 연구에서는 디알릴테레프탈레이트와 1,3-부탄디올을 단량체조 하여 알릴 에스터 예비 중합체를 합성하고, 점토를 이용하여 층간 삽입법과 직접 중합법으로 나노복합재를 제조하여 점토의 함량, 경화조건, 점토의 혼합 방법에 따른 특성을 분석하였다. 실리케이트 층간 거리는, 30B-점토를 이용하여 직접 중합법으로 제조하였을 때, 40$\AA$ 이상으로 가장 넓게 나타났다. 이는 유기화제의 작용기 (-OH)와 단량체가 실리케이트의 층 사이에서 에스터 교환 반응을 일으켜, 층간 거리가 증가하였기 때문이다. 또한 기계적 특성과 열적 특성 확인으로 점토의 분산 정도가 복합재의 물성 향상의 중요한 인자임을 확인할 수 있었다.

• Restorative Dentistry and Endodontics
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• 제29권4호
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• pp.399-408
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• 2004

Odontoblasts are responsible for the formation and maintenance of dentin. They are known to synthesize unique gene products including dentin sialophosphoprotein (DSPP). Another unique genes of the cells remain unclear. OD314 was isolated from the odontoblasts/pulp cells of rats and partially characterized as an odontoblast-enriched gene (Dey et al., 2001). This study aimed to elucidate the biological function of OD314, relating to odontoblast differentiation and dentinogenesis. After determining the open reading frame (ORP) of OD314 by transient transfection analysis using green fluorescent protein (GPP) expression vector, mRNA in-situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western analysis were performed. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were expressed in odontoblasts of developing coronal and root pulp. 2. OD314 was a novel protein encoding 154 amino acids, and the protein was mainly expressed in cytoplasm by transient transfection analysis. 3. Mineralized nodules were associated with multilayer cell nodules in the culture of human dental pulp cells and first detected from day 21 using alizarin-red S staining. 4. In RT-PCR analysis, OD314, osteocalcin (OC) and DSPP strongly expressed throughout 28 days of culture. Whereas, osteonectin (ON) mRNA expression stayed low up to day 14, and then gradually decreased from day 21. 5. Western blots showed an approximately 17 kDa band. OD314 protein was expressed from the start of culture and then increased greatly from day 21. In conclusion, OD314 is considered as an odontoblast-enriched gene and may play important roles in odontoblast differentiation and dentin mineralization.

• 한국수산과학회지
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• 제48권1호
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• pp.26-35
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• 2015

This study compared the meat quality of sea rainbow trout Oncorhynchus mykiss frame muscle (S-FR-TY) and fillet (S-FI-TY) with the frame muscle of freshwater rainbow trout (F-FR). There was a difference of < 1% in the proximate composition of S-FR-TY vs. S-FI-TY, and of S-FR-TY vs. F-FR. The Hunter a value of F-FR-TY was lower compared with that of F-FR cultured in Jecheon (F-FR-JC), but higher than that of F-FR cultured in Pyeongtaek (F-FR-PT). However, no difference in the Hunter a value of S-FR-TY compared with any other F-FR was observed. The odor intensity of S-FR-TY was lower compared with that of F-FR, while the taste of S-FR-TY was milder than that of S-FI-TY, but stronger than that of F-FR. Although its total amino acid content was lower, the essential amino acid content of S-FR-TY was higher compared with S-FI-TY; the potassium content of S-FR-TY was also significantly greater. S-FR-TY contained high levels of nutritional and functional components, such as anserine, lysine, threonine, and eicosapentaenoic and docosahexaenoic acid. These results suggest that S-FR-TY could be used as a resource of canned food or fish jerky for children.

• Journal of Microbiology and Biotechnology
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• 제25권1호
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• pp.18-25
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• 2015

To understand the metabolism of flavonoid rhamnoglycosides by human intestinal microbiota, we measured the metabolic activity of rutin and poncirin (distributed in many functional foods and herbal medicine) by 100 human stool specimens. The average α-L-rhamnosidase activities on the p-nitrophenyl-α-L-rhamnopyranoside, rutin, and poncirin subtrates were 0.10 ± 0.07, 0.25 ± 0.08, and 0.15 ± 0.09 pmol/min/mg, respectively. To investigate the enzymatic properties, α-L-rhamnosidase-producing bacteria were isolated from the specimens, and the α-L-rhamnosidase gene was cloned from a selected organism, Bifidobacterium dentium, and expressed in E. coli. The cloned α-L-rhamnosidase gene contained a 2,673 bp sequcence encoding 890 amino acid residues. The cloned gene was expressed using the pET 26b(+) vector in E. coli BL21, and the expressed enzyme was purified using Ni²⁺-NTA and Q-HP column chromatography. The specific activity of the purified α-L-rhamnosidase was 23.3 µmol/min/mg. Of the tested natural product constituents, the cloned α-L-rhamnosidase hydrolyzed rutin most potently, followed by poncirin, naringin, and ginsenoside Re. However, it was unable to hydrolyze quercitrin. This is the first report describing the cloning, expression, and characterization of α-L-rhamnosidase, a flavonoid rhamnoglycosidemetabolizing enzyme, from bifidobacteria. Based on these findings, the α-L-rhamnosidase of intestinal bacteria such as B. dentium seem to be more effective in hydrolyzing (1 →6) bonds than (1 →2) bonds of rhamnoglycosides, and may play an important role in the metabolism and pharmacological effect of rhamnoglycosides.

• Journal of Microbiology and Biotechnology
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• 제28권7호
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• pp.1185-1198
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• 2018

The nucleosomal organization of chromatin using histone proteins is a fundamental and ubiquitous feature of eukaryotic nuclei, with the major exception of dinoflagellates. Although a number of recent genomic and transcriptomic analyses have detected numerous histone genes in dinoflagellates, little is known about their expression. Here in, we aimed to investigate the expression pattern of histone genes under nutritional stress, and an attempt was made to detect histone expression at the protein level in Alexandrium pacificum. The presence of histones at the mRNA level was confirmed in this study by the amplification, cloning, and sequencing of 10 different genes. Relative expression profiling of these genes under different growth conditions was determined with real-time PCR and revealed considerable levels of histone transcription in nutritionally stressed cells. We were unable to detect the expression of histones at the protein level even after immunodetection and analysis using mass spectrometry, although a histone-like protein was detected as a major nuclear component. A. pacificum expresses multiple variants of histone, and protein sequences revealed both conservation and divergence with respect to other eukaryotes. We concluded that A. pacificum maintained an active transcription of histone genes within the cell, and enhanced expression of histone genes in nutritional stress strongly suggest that histones have functional significance in dinoflagellates, although expression at the protein level was below our current detection limits, which suggests a limited role of histones in DNA packaging. Finally, the plausible regulation of histone expression at the gene and protein levels in A. pacificum is discussed.

• Journal of Microbiology and Biotechnology
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• 제30권5호
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• pp.726-732
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• 2020

Indonesian kefir grains are potential sources of lactic acid bacteria (LAB) that may act as probiotics with specific functional properties. In this study we explored the potential of the probiotic and cholesterol-lowering effect of LAB isolated from Indonesian kefir grains obtained from Bogor, Bandung, Jakarta, and Yogyakarta. The results revealed that 10 isolates showed considerable survivability at low pH and bile salt with total cell reduction of ~3 log colony-forming units per milliliter after exposure to pH 2.5 and 0.5% (w/v) bile salt for 1 and 3 h, respectively. All strains exhibited strong antimicrobial activities against pathogenic bacteria and were sensitive to a wide spectrum of antibiotics but exhibited weak bile salt hydrolase activity. Identification based on 16S RNA suggested that nine isolates were Lactobacillus kefiri and one was Lactobacillus rhamnosus. The ability of the isolates to reduce cholesterol from the media varied, ranging from 22.08% to 68.75% with the highest reduction shown by L. kefiri JK17. The ability to remove cholesterol from the media decreased greatly in resting and dead cells, ranging from 14.58% to 22.08% in resting cells and from 7.89% to 18.17% in dead cells. It can be concluded that Indonesian kefir grains contain LAB potentially acting as probiotics capable of reducing cholesterol. The cholesterol-lowering effect especially occurs when the cells are metabolically active.