Elicitins, proteinaceous elicitors secreted from Oomycetes fungi (Phytophthora spp. and Pythium spp.), have been known as inducer of hypersensitive response (HR) in incompatible interactions between plant and pathogens. Five elicitins among many Korean Phytophthora species caused the reactions of distal HR in radish, chinese cabbage and some hot pepper cultivars, but not in cucumber and tomato. Because the isolation of elicitin from Phytophthora cambivora hasn't been reported yet, we have purified a cambivorein, a new member of the elicitin family, from the culture filtrate of Phytophtilora cambivora (KACC 40160) by using FPLC (Fast Protein Liquid Chromatography, AKTA) with sepharose S and Sephacryl HR columns. We confirmed that it induces necrosis activities in some hot pepper cultivars and its molecular weight is about 10 KDa by Tricine-SDS-PAGE. Comparison of amino acid sequences of its N-terminal ends also informed the identification of Iysine at the 13th position, which is characteristic of a kind of basic elicitin isoform $({\beta}-isoform)$. It Also showed that our elicitin is not identical with N-terminal sequences of many elicitins reported from Phytophthora spp..
Microorganism (strain DF 218) producing thermostable pretense was isolated from Korean soil and compost. It was Gram-positive, rod-shaped, aerobic, and spore-forming with yellowish white colony color, Temperature range for growth at pH 6.5 was $30-65^{\circ}C$, with optimum growth at $60^{\circ}C$. pH range for growth at $60^{\circ}C$ was 5-7 with optimum of 6.5, which indicates strain DF 218 to be thermophilic. The 16S rDNA sequence of strain DF 218 had 95% sequence similarity with that of Bacillus flexus. Based on physiological properties and phylogenetic analysis, we proposed the isolated strain as Bacillus sp. DF 218. Pretense was produced aerobically at $60^{\circ}C$ for 32 hr in a medium (pH 6.5) containing 1% each trypton, glucose, and NaCl. Its molecular weight was estimated as 61 kDa, with optimum temperature and pH of $60^{\circ}C$ and 7.5, respectively.
A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80℃ in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50℃ and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni2+, Mn2+, and Cu2+ to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The Km and Vmax values were estimated to be 35.7 mg/ml, and 5 × 104 U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.
Park, Jong-Hoon;Oh, Keum-Soon;Kang, Kil-Jin;Kim, Kwan
Applied Biological Chemistry
/
v.43
no.4
/
pp.241-246
/
2000
In order to investigate structural properties of Chindo black rice(grown in Chindo, Chonnam) starch and its amylopectin, Chindo black rice was investigated in comparison to Shinsun waxy rice. The maximum absorbance wave and intrinsic viscosity of Chindo black rice starch and Shinsun waxy rice starch were 523 nm, 521 nm and 183 ml/g, 178 ml/g, respectively. ${\beta}-amylolysis$ limit(%) of Chindo black rice and Shinsun waxy rice starch were 62.8% and 60.3%, respectively. Chindo black rice was determined to be a waxy rice due to the results of iodine reaction and elution profile on Sephroce CL-2B. The chain of amylopectins in Chindo black rice distributed fraction 1$(F_1)$ of above degree of polymerization$({\overline{DP}})$ 55, fraction 2$(F_2)$ of ${\overline{DP}}$40{\sim}50$ and fraction 3$(F_3)$ of ${\overline{DP}}$15{\sim}20$, and the ratio of $F_3$ to $F_2$ for Chindo black rice was higher than that for Shinsun waxy rice. The super long chain of amylopectin in Chindo black rice was consisted much more than that of Shinsun waxy rice. ${\beta}-limit$ dextrins in Chindo black rice amylopectin distributed $F_1$ of above ${\overline{DP}} 55, $F_2$ of ${\overline{DP}}$30{\sim}45$ and $F_3$ of ${\overline{DP}}$10{\sim}20$. Little difference was shown between elution patterns of the pullulanase treated ${\beta}-limit$ dextrins of Chindo black rice amylopectin and Shinsun waxy rice amylopectin. These results suggest that Chindo black rice starch was similar to Shinsun waxy rice starch.
Human neutrophil cathepsin-G, which has been known as one of the active enzymes causing inflammatory diseases, was purified by two steps procedure involving one size exclusion (Ultorogel AcA54) and one ion exchange (CM-Sephadex) chromatography. Purified HNCGs were cross-reacted with Anti-HNCathepsin-G antibodies which were radised in rabbits and purified by cathepsin-G labeled Sepharose 4B affinity chromatography. HNCGs were effectively inhibited by NSAIDs including phenylbutazone, sulindac, oxyphenbutazone, salicylic acid and salicyluric acid. $IC_{50}_s$ of these drugs for inhibition of Cathepsin G were 0.3-0.8 mM. Other NSAIDs including aspirin showed little or no inhibition effect on the activity of Cathepsin G. These results strongly indicated that NSAIDs which showed inhibition effect on the activity of HNCGs possibly be at least a part of mechanism of action which might be related to direct inhibition of cathepsin G at the tissue destruction sites beside of their known mechanism of action as an anticyclo-oxygenase in treatment of inflammatory diseases. Lipid soluble component of Korean Red Ginseng which was known as an anti-inflammatory agent inhibited HNCGs strongly, but no other fractions did inhibited HNCGs. Antibiotics including novobiosin and rifamycin showed some inhibition effect on HNCGs, i. e.., $IC_{50}$ of these drugs were 2.6 mM and 1.5 mM respectively, and other antibiotics including penicillin G showed no or negligible inhibition effect on the activity of HNCGs. However. tetracyclines inhibited HNCGs very effectively at the concentration of therapeutic range. The inhibition effect of the activity of HNCGs by tetracycline are not related to the N-dimethyl radical on the 4 position of the tetracycline molecule. Furthermore, N-dedimethylated tetracyclines may have beneficial effect for long term treatment of chronic inflammatory diseases without developing any drug resistance to microorganisms.
Kim, Young-Ho;Joo, Ye-Jin;Jung, Hye-Mi;Seo, Un-Kyo
The Journal of Korean Medicine
/
v.29
no.3
/
pp.63-75
/
2008
Objectives: In the present study, the author intended to investigate whether Gamijinhae-tang (Jiaweizhenke-tang) (GJHT) significantly affects both contractility of tracheal smooth muscle and mucin secretion from airway epithelial cells. Materials and Methods: Effect of GJHT on contractility of isolated tracheal smooth muscle of rabbit was investigated. Confluent hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled with 3H-glucosamine for 24 hrs and chased for 30 min in the presence of GJHT to assess the effect of the agent on 3H-mucin secretion. At the same time, confluent NCI-H292 cells were chased for 30 min in the presence of GJHT to assess the effect of the agent on MUC5AC secretion by ELISA. Total elution profiles of control spent media and treatment sample (radioactive mucin) through Sepharose CL-4B column were analyzed. Also, effect of the agent on MUC5AC gene expression in cultured NCI-H292 cells was investigated. Possible cytotoxicities of the agent were assessed by measuring both lactate dehydrogenase (LDH) release from HTSE cells and examining the rate of survival and proliferation of NCI-H292 cells. Results: (1) GJHT inhibited Ach-induced contraction of isolated tracheal smooth muscle; (2) GJHT significantly increased mucin secretion from cultured HTSE cells. However, it did not affect MUC5AC secretion from NCI-H292 cells, only chiefly affecting the 'mucin' secretion; (3) GJHT did not significantly affect the expression levels of MUC5AC gene in cultured NCI-H292 cells; (4) GJHT did not significantly inhibit the survival and proliferation of NCI-H292 cells. However, it slightly increased LDH release from HTSE cells. Conclusion: The author suggests that effects of GJHT with their components should be further investigated and it is valuable to find, from oriental medical prescriptions, novel agents which might regulate mucin secretion from airway epithelial cells.
The objective of this study was to control Kimchi fermentation using pH sensitive bacteriocin entrapping liposome(bacteriocin-liposome). The liposomes were prepared by the reverse-phase evaporation method from a mixture of DPPC(dipalmitoyl phosphatidylcholine, DPPE(dipalmitoyl phosphatidylethanolamine), DOPC(dioleoyl phosphatidylcholine) and cholesterol in a molar ration of 4:2:1:4. The bacteriocin-liposome was disruptured at pH 4 of buffer and was stable at alkaline pHs(6 and 7). Irrespective of the addition of the bacteriocin-liposomes, the pH of every Kimchi sample decreased to 5 during 5 days storage at 5$^{\circ}C$. Kimchi samples treated with bacteriocin-liposomes maintained pH 4 or higher, while Kimchi samples not treated with bacteriocin-liposomes exhibited pH 3.58 or lower. In general, the pH of Kimchi samples stored at 20$^{\circ}C$ decreased faster, compared to that of Kimchi samples stored at 5$^{\circ}C$. The pH of Kimchi samples treated with the bacteriocin-liposomes was 3.9 during 90 days storage, while that of the samples not treated with the bacteriocin-liposomes was 3.68 and 3.32 during 30 days and 90 days storages, respectively. Lactic acid bacteria in Kimchi treated with the bacteriocin-liposome grew relatively slow at 5$^{\circ}C$. The viable cell number of lactic acid bacteria increased up to 4${\times}$10$\^$7/ cells/ml and then decreased to 8${\times}$10$\^$6/ cells/ml during 90 days storage at 5$^{\circ}C$.
A thermophilic microorganism, strain JE 375, which produces a thermostable protease, was isolated from soil and compost in Korea. This gram-positive, rod-shaped, catalase positive, motility positive, and hemolysis ${\beta}$ containing organism was implicated in glucose fermentation, mannitol fermentation, xylose oxidation, aerobic activity and spore formation. The color of the colony was yellowish white. The temperature range for growth at pH 6.5 was between 55 and $70^{\circ}C$, with an optimum growth temperature of $65^{\circ}C$. This result confirmed the strain JE 375 as a thermophilic microorganism. The enzyme was produced aerobically at $65^{\circ}C$ during 20 hr in a medium (pH 6.5) containing 1% trypton. 1% maltose, 0.5% yeast extract and 1% NaCl. The 16S rDNA of strain JE 375 had 97.6% sequence similarity with the 16S rDNA of Bacillus caldoxyloyticus. On the basis of biochemical and physiological properties and phylogenetic analysis, we named the isolated strain as Bacillus sp. JE 375. The thermostable protease from Bacillus sp. JE 375 had been partially purified and characterized. The molecular weight of the enzyme was deduced from SDS-PAGE and gel chromatography as 55 kDa and its optimal temperature was $60^{\circ}C$. The enzyme showed its highest activity at pH 7.5 and was stable from pH 7.0 to 8.0.
Previously, we discovered a new MMP-2 isoform GA110, of which appearance in human follicular fluid(FF) and serum was increased by EDTA. The present study was conducted to investigate how GAI 10 can appear by EDTA. To examine possible involvement of protein disulfide isomerase(PDI), an enzyme responsible for the dimerization of protein via disulfide formation, effect of PDI inhibitor on the appearance of GA110 by EDTA was investigated. When PDI inhibitor added to FF before EDTA treatment, the gelatinolytic activity of GA110 was abolished in a concentration dependent manner. By contrast, the activity of 72 kDa gelatinase increased. However, the PDI inhibitor added to FF after EDTA treatment, the gelatinolytic activity of GA110 was unaffected. To find out the nature of the enzyme which converts 72 kDa gelatinase into GAI 10, chromatographic separation method of FF proteins was done. Using hydroxyapatite column, fractions rich in 72 kDa gelatinase were isolated and pooled. By using this pool as substrate for the 72 kDa converting enzyme, protein fractions containing the converting activity were obtained from chromatographic separation of FF onto glutathione sepharose fast flow column. When immunoblotting was performed on this enzymatically active protein fractions against polyclonal anti-PDI antibody, distinct immunoreactivity was observed, although appeared in smaller molecular weight region. Based on these observations, it is suggested that the appearance of GAI 10 in FF by EDTA treatment could be due to an activation of PDI-like enzyme, which dimerizes 72 kDa gelatinase into GAI 10 via the formation of disulfide bond between molecules.
The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.
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