• Parasites, Hosts and Diseases
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• v.56 no.1
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• pp.25-32
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• 2018

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris-EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was $40parasites/{\mu}l$ for P. falciparum and $35.2parasites/{\mu}l$ for P. vivax, whereas for Sn-PCR the limit of detection was $1.6parasites/{\mu}l$ for P. falciparum and $1.4parasites/{\mu}l$ for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

• BMB Reports
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• v.34 no.2
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• pp.109-113
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• 2001

The Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). The three-dimensional structure of SmaPI was calculated by computer modeling using the structure complex between SMP and the Erwinia chrysanthemi inhibitor as a template. Based on this model structure, the substitution of the amino acid residues, Ala4, Leu-5, Pro-6, and Thr-7, were located at the hinge region of the N-terminal segment by site-directed mutagenesis. Although the A4R and T7A mutant SmaPIs showed a nearly full inhibitory activity, the inhibitory activity of SmaPI decreased significantly when the Leu-5 was converted to Ala, Gly, Ile, or Val. Surprisingly, the L5I and L5V mutant SmaPIs showed less inhibitory activities than the L5A mutant. From these results, we suggested that the orientations and positions of respective aliphatic groups in the side-chain of position 5 mainly affected the inhibitory activity of SmaPI. The overall side-chain hydrophobicity was only slightly affected. The side-chain of the Leu-5 residue contributed approximately 0.79 kcal/mol out of 8.44 kcal/mol to the binding of SmaPI with SMP The inhibitory activities of P6A and F6G were also severely decreased. The Pro-6 may have a critical role in maintaining the strict conformation of the N-terminal portion that may be important in the inhibitory activity of SmaPI. In conclusion, Leu-5 and Pro-6 have crucial roles in the inhibitory function of SmaPI toward SMP.

• BMB Reports
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• v.30 no.1
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• pp.26-32
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• 1997

Of all HLA class I molecules, HLA-C gene products are most poorly understood because they express at a low level on the cell surface compared to HLA-A and -B. In order to identify serologically detectable and undetectable HLA-C antigens, we have established a DNA-based tissue typing method for the HLA-C locus by PCR-SSP (polymerase chain reaction-sequence specific primers). Genomic DNA prepared from Iymphoblastoid 21 B-cell lines and 120 Korean individuals by proteinase K digestion and pheno/chloroform extractions have been typed by PCR-SSP (23 primer mixes were used). The PCR-SSP results of control cell lines were discrepant from serology in 1 case among 21 cases: Cw6 which was negative by serology but positive by PCR-SSP (cell line: MANIKA). Twenty four HLA-Cw "blank" antigens among fifty Korean individuals were completely determined by PCR-SSP DNA typing. HLA-Cw*0101 (15.3%), Cw*1401 (12.3%) and Cw*0701 (11.7%) alleles were frequently found in 120 Korean individual samples. In conclusion. the high level of discrimination for HLA-C alleles may prove useful and informative in the study of transplant survival, and identify the importance of allelic differences, not readily detectable by serology, on host and donor compatibility.

• Parasites, Hosts and Diseases
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• v.55 no.2
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• pp.115-120
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• 2017

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.

• Natural Product Sciences
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• v.11 no.4
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• pp.207-212
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• 2005

Tetragonia tetragonoides (Aizoaceae) has been known as an anti-cancer agent. The activation of proteinase-activated receptor-2 (PAR-2) by trypsin appears to play a role in inflammation. In the present study, we examined the inhibitory effects of Tetragonia tetragonoides water extract (TTWE) on the production of tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$ and tryptase in trypsin-stimulated human leukemic mast cells (HMC-1) expressing PAR-2. HMC-1 cells were stimulated with trypsin in the presence or absence of TTWE (10, 100, and $1000\;{\mu}g/ml$). The level of $TNF-{\alpha}$ secretion from HMC-1 cells was measured by enzyme-linked immunosorbent assay (ELISA). $TNF-{\alpha}$ and tryptase mRNA expression were examined by reverse transcription-PCR. Also, extracellular signal-regulated kinese (ERK) activation was assessed by Western blot analysis. Trypsin activity was measured using the substrate Bz-DL-Arg-p-nitroanilide (BAPNA). It was observed that $TNF-{\alpha}$ secretion, tryptase mRNA and $TNF-{\alpha}$ mRNA expression in trypsin-stimulated HMC-1 cells were inhibited by pretreatment of TTWE ($1000\;{\mu}g/ml$). Furthermore, the pretreatment of TTWE ($1000\;{\mu}g/ml$) resulted in the reduction of ERK phosphorylation and trypsin activity. These results suggest hat TTWE might have the inhibitory effects on the PAR-2-dependent inflammation processes and it is likely to function as PAR-2 antagonist.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1493-1501
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• 2015

A Bacillus species, EMD4, with strong antibacterial activity was isolated from ganjang (soy sauce) and identified as B. subtilis. B. subtilis EMD4 strongly inhibited the growth of B. cereus ATCC14579 and B. thuringiensis ATCC33679. The antibacterial activity was stable at pH 3-9 but inactive at pH 10 and above. The activity was fully retained after 15 min at 80℃ but reduced by 50% after 15 min at 90℃. The activity was completely destroyed by proteinase K and protease treatment, indicating its proteinaceous nature. The bacteriocin (BacEMD4) was partially purified from culture supernatant by ammonium sulfate precipitation, and Q-Sepharose and Sephadex G-50 column chromatographies. The specific activity was increased from 769.2 AU/mg protein to 8,347.8 AU/mg protein and the final yield was 12.6%. The size of BacEMD4 was determined to be 3.5 kDa by Tricine SDS-PAGE. The N-terminal amino acid sequence was similar with that of Subtilosin A. Nucleotide sequencing of the cloned gene confirmed that BacEMD4 was Subtilosin A. BacEMD4 showed bactericidal activity against B. cereus ATCC14579.

• Journal of the Korean Applied Science and Technology
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• v.34 no.3
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• pp.643-649
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• 2017

Biodegradable enzymes such as lipase and proteinase can hydrolyze not only fatty acid esters and triglycerides, but also aliphatic polyesters. We measured the biodegradability that biodegradable enzymes have an important role in the degradation of natural aliphatic poly material such as PLA, corn starch, and polyethylene glycol in the natural environment. However, we investigated on the biodegradability of PLA, PLA and Polyethylene acrylate blended, and PLAcoPolyethylene polymerized with PLA graft copolymer Polyethylene glycol acrylate. When prepared biodegradable polymers. the Mechanical properties of them were measured on Biodegradability, thermal properties, real time in-situ electrical monitoring of polymers resin. Therefore BOD and biodegradation of PLAcoPolyethylene was graft copolymerized with PLA and polyethylene acrylate were measured at a lower rate than the other samples.

• The Korean Journal of Mycology
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• v.25 no.4 s.83
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• pp.320-329
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• 1997

Botrytis cinerea isolates obtained from infected plants of cucumber, tomato, and strawberry were divided into three groups (sporing, sclerotial, and mycelial types). Of which sclerotial types were the major group. There were no correlations between morphological phenotypes and responses to benzimidazole and dicarboximide fungicides. External structure of conidia of three phenotypes by scanning electron microscope was the same with verrucose surface. Mycelial type was the most virulent on fruits of eggplants. Comparative tests were carried out to examine correlations between the virulence and production of fungal enzymes such as phenol oxidases, pectin methyl esterases (PME), amylases, cellulases, ureases, ${\beta}-glucosidases$, and proteinases. There was no correlation among the phenotypes in production of phenol oxidases and ${\beta}-glucosidases$. However, there were significantly different from each other in PME, amylase, cellulase, urease, and protease activity.

• KSBB Journal
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• v.26 no.6
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• pp.513-516
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• 2011

The occurrence of dental caries is mainly associated with oral pathogens, especially cariogenic Streptococcus mutans. The aim of this study was to isolate and characterize lactic acid bacterium showing inhibitory activity against cariogenic Streptococcus mutans. As results, an isolate with strong inhibitory activity was obtained from Kimchi and it was identified as Lactobacillus sakei by API and 16S rRNA gene analyses. This strain secreted an inhibitory compound in cell growth medium and the activity of the compound was completely disappeared by proteinase K revealing the fact that the compound is proteinous substance, bacteriocin. Optimal culture condition for bacteriocin production by Lb. sakei K-7 was at pH 7.5 and $37^{\circ}C$ for 18 h. Oral administration of this isolate may give anticariogenic and probiotic effects on hosts.

• BMB Reports
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• v.32 no.1
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• pp.86-91
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• 1999

The sprT gene encoding Streptomyces griseus trypsin (SGT) was introduced into Streptomyces lividans TK24 and Streptomyces lividans 1326 to study which strain would be better to overexpress the extracellular proteinase. Various media with different compositions were also used to maximize the productivity of SGT in heterologous hosts. The SGT productivity was best when the transformants of S. lividans TK24 and 1326 were cultivated in R2YE medium, and their relative trypsin activity of the culture broth measured with an artificial chromogenic substrate, N-${\alpha}$-benzoyl-DL-arginine-${\rho}$-nitroanilide, were 382 units/ml and 221 units/ml, respectively. They produced high levels of SGT in GYE medium but relatively lower than those in R2YE medium, and negligible amount of SGT was produced in Ferm, RASF, LIVID, and NDSK media. Considering non-SGT associated activity in Pronase powder, it was estimated that the transformant of S. lividans TK24 can produce SGT in R2YE 3.5 times more than the amount by S. griseus 10137 from which the sprT gene had been originated. The growth of S. lividans reached the maximum level of cell mass at 5 d of culture, but SGT production started in the stationary phase of cell growth and kept increasing until the ninth day of culture in R2YE medium, but in GYE media the productivity reached at the maximum level at 7 d of cultivation.