• 대한임상검사과학회지
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• 제39권3호
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• pp.147-150
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• 2007

Most expressed HLA (human leukocyte antigen) loci exhibit a remarkable degree of allelic polymorphism, which derives from sequence differences predominantly localized to discrete hypervariable regions of the amino terminal domain of the molecule. In this study, the HLA-B genotypes were determined in twenty students unrelated koreans using the PCR-SSP (polymerase chain reaction-sequence specific primer) technique. Several specific primer pairs in assigning the HLA-B gene were used ($B^{\ast}4001/4007$, $B^{\ast}4901/5001/4501$, $B^{\ast}3701$, $B^{\ast}5801$). The results of PCR-SSP, the HLA-B3701 primer was detected one (5%), the $HLA-B^{\ast}5801$ were detected four (20%), the $HLA-B^{\ast}4001/4007$ were detected nineteen (95%) and the $HLA-B^{\ast}4901/5001/4501$ were detected twenty. This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-B genotypes. Moreover, these results genotype frequency of the HLA-B gene could be useful for database study before being applied to individual identification and transplantation immunity.

• Archives of Pharmacal Research
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• 제24권4호
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• pp.316-322
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• 2001

Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library copy of Citrobacter freundii, subcloned into the vector pGEM3Zf(-) and sequenced. Sequencing revealed two contiguous open reading frames (ORF1 and ORF2) on the same strand and based on amino acid sequence homologyl they were designated as astA and dsbA, respectively. The amino acid sequence of astA deduced from C. freundii was highly similar to that of the Salmonella typhimurium, Enterobacter amnigenus, Klebsiella, Pseudomonas putida, and Campylobacter jejuni, encoded by the astA genes. However, the ASST activity assay revealed different acceptor specificities. Using p-nitrophenyl sulfate (PNS) as a donor substrate, $\alpha$-naphthol was found to be the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine and tyrosine.

• Journal of Microbiology and Biotechnology
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• 제24권7호
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• pp.998-1003
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• 2014

Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and ${\alpha}$-ketoglutarate to oxaloacetate and glutamate. It plays a key role in amino acid metabolism and has been widely recommended as a biomarker of liver and heart damage. Our study aimed to evaluate the extensive preparation of AST and its application in quality control in clinical laboratories. We describe a scheme to express and purify the 6His-AST fusion protein. An optimized sequence coding AST was synthesized and transformed into Escherichia coli BL21 (DE3) strain for protein expression. Ideally, the fusion protein has a volumetric productivity achieving 900 mg/l cultures. After affinity chromatography, the enzyme activity of purified AST reached 150,000 U/L. Commutability assessment between the engineered AST and standard AST from Roche suggested that the engineered AST was the better candidate for the reference material. Moreover, the AST showed high stability during long-term storage at $-20^{\circ}C$. In conclusion, the highly soluble 6His-tagged AST can become a convenient tool for supplying a much better and cheaper standard or reference material for the clinical laboratory.

• BMB Reports
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• 제44권9호
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• pp.578-583
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• 2011

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are biochemical markers used to test for liver diseases. Copy number variation (CNV) plays an important role in determining complex traits and is an emerging area in the study various diseases. We performed a genome-wide association study with liver function biomarkers AST and ALT in 407 unrelated Koreans. We assayed the genome-wide variations on an Affymetrix Genome-Wide 6.0 array, and CNVs were analyzed using HelixTree. Using single linear regression, 32 and 42 CNVs showed significance for AST and ALT, respectively (P value < 0.05). We compared CNV-based genes between the current study (KARE2; AST-140, ALT-172) and KARE1 (AST-1885, ALT-773) using NetBox. Results showed 9 genes (CIDEB, DFFA, PSMA3, PSMC5, PSMC6, PSMD12, PSMF1, SDC4, and SIAH1) were overlapped for AST, but no overlapped genes were found for ALT. Functional gene annotation analysis shown the proteasome pathway, Wnt signaling pathway, programmed cell death, and protein binding.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.571-575
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• 2003

A marine aerobic Gram-negative bacterium, Pseudoalteromonas carrageenovora, has been blown to hydrolyze carrageenans, the sulfated galactans of red algae, and to desulfate oligo kappa-carrageenans. Recently, the gene encoding arylsulfatase (aryl-sulfate sulfohydrolase, E.C.3.1.6.1) of A. carrageenovora was cloned and the nucleotide sequence was reported. Enzymatic hydrolysis of sulfate groups in agaropectin simplifies the process of agarose preparation. In order to overproduce the enzyme, the arylsulfatase gene (astA, 984 bp ORF) from P. carrageenovora genome was amplified by PCR and subcloned into the pET21a vector. When the constructed plasmid pAST-A1 was introduced into E, coli BL21(DE3), the transformant on LB plate containing IPTG showed the hydrolyzing activity for p-nitrophenyl sulfate. Most of arylsulfatase activity was found in the cell lysate, but at $50\;{\sim}\;5000\;{\mu}M$ IPTG concentration the activity was found both in the culture supernatant and the cell lysate. The molecular weight of the recombinant enzyme was estimated to be 34 kDa by SDS-PAGE.

• Applied Biological Chemistry
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• 제39권3호
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• pp.195-199
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• 1996

$crp^{\ast}$ 유전자가 도입된 대장균 MK2001($crp^{{\ast}1}$}, cya::km)을 숙주로 사용하여 mal 유전자 발현을 촉진시키는 유전자의 하나인 sfs1(sugar fermentation stimulation)의 구조해석 결과에 의하면, 잠정적인 sfs1의 promoter 영역에는 CRP 단백질과의 결합영역으로 보이는 염기배열이 존재하였다. 본 실험에서는 sfs1 유전자의 cAMP-CRP에 의한 발현 조절을 확인하고자, lacZ 와의 융합 유전자를 작성하였다. 작성된 융합 유전자는 cya 결손주인 Tp2010에서 cAMP의 첨가에 의해 ${\beta}-galactosidase$ 활성이 크게 증가하였으며, Western blotting의 실험에서도 같은 결과를 나타냈다. in vivo에서 발현이 확인된 전사산물은 cAMP에 의해 전사 촉진이 일어났으며, CRP의 결합부위로 예상되는 DNA 영역은 cAMP가 존재하면 CRP 단백질과 결합하는 특성을 나타내었다. 이상의 결과로 보아, sfs1 유전자의 발현은 UMP-CRP에 의한 전사촉진 현상을 받는 것으로 나타났다.

• 대한임상검사과학회지
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• 제37권3호
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• pp.139-142
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• 2005

Most expressed HLA(human leukocyte antigen) loci exhibit a remarkable degree of allelic polymorphism, which is derived from sequenceing differences predominantly localized to discrete hypervariable regions of the amino-terminal domain of the molecule. In this study, the HLA-DRB1 genotypes were determined in twenty students using the PCR-SSP (polymerase chain reaction-sequence specific primer) technique. Two specific primer pairs in assigning the DRB1 gene were used. The results of PCR-SSP, the $HLA-DRB1^{\ast}0101$ primer detected nine and $HLA-DRB1^{\ast}1501$ primer detected three people. This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-DRB1 genotypes. Moreover, these genotype frequency results of the HLA DRB1 gene could be useful for database study before being applied to individual identification and transplantation immunity.

• 대한약학회:학술대회논문집
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• 대한약학회 2000년도 춘계총회 및 학술대회
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• pp.156.2-156.2
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• 2000

• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.126-133
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• 2006

Thioacetamide (TA) is well known hepatotoxic and hepatocarcinogenic agent. TA also diminishes the contents of hepatic cytochrome P450 and inhibits the enzyme activity of the hepatic mixed function oxidases. TA metabolite, thioacetamide-s-oxide, is further transformed into a still unknown highly reactive metabolite that binds to macromolecules. In this study, we focused on TA-induced gene expression at hepatotoxic dose. Mice were exposed to two levels (5 mg/kg or 50 mg/kg i.p.) of TA, sampled at 6 or 24 h, and hepatic gene expression levels were determined to evaluate dose and time dependent changes. We evaluated hepatotoxicity by serum AST and ALT level and histopathological observation. Mean serum activities of the liver leakage enzymes, AST and ALT, were slightly increased compare to control. H & E and PAS evaluation of stained liver sections revealed TA-associated histopathological finding in mice. Centrilobular eosinophilic degeneration was observed at high dose-treated mice group. Hepatic gene expression was analyzed by QT clustering. Clustering of high dose-treated samples with TA-suggests that gene expressional changes could be associated from toxicity as measured by traditional biomarkers in this acute study.

• Molecular & Cellular Toxicology
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• 제1권4호
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• pp.275-280
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• 2005

Carbon tetrachloride ($CCl_4$) is well known hepatotoxicant. Its overdose cause severe centrilobular hepatic necrosis in human and experimental animals. We administered $CCl_{4}$ at low (0.2 mL/kg p.o.) and high (2 mL/kg p.o.) doses to mice. Mice were sacrificed at 24 h after administration. We evaluated liver toxicity by serum AST and ALT level and by microscopic observation. Using cDNA chip, we conducted gene expression analysis in liver. Mean serum activities of the hepatocellular leakage enzymes, ALT and AST, were significantly increased compare to control, respectively, in the low and high dose groups. H&E evaluation of stained liver sections revealed $CCl_{4}-related$ histopathological findings in mice. Moderate centrilobular hepatocellular necrosis was present in all $CCl_{4}$ treated mice. We found that gene expression pattern was very similar between low and high dose group. However, some stress related genes were differently expressed. These results could be a molecular signature for the degree of liver injury. Our data suggest that the degree of severity could be figure out by gene expression profiling.