• The Korean Journal of Mycology
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• v.13 no.1
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• pp.1-10
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• 1985

This experiment was undertaken to investigate proper conditions for protoplast isolation from strains of Agaricus bisporus, Flammulina velutipes, Lentinus edodes, Pleurotus florida, Pleurotus ostreatus, Pleurotus sajor-caju and Volvariella volvacea. Combination of ${\beta}-D-glucanase$, novozym 234 and snail enzyme with 0.6M $MgSO_4$ in 0.05M Na-maleate buffer, pH 5.8 was the most effective for isolation of protoplasts. High yields of protoplasts were obtained from $4{\sim}5$ days old mycelia of P. florida and P. ostreatus on mushroom complete agar medium. Protoplasts were also obtained in good yield from other fungi but A. bisporus and V. volvacea. In the latter two cases protoplast release was observed; however, the yield was much lower than those of the other fungi.

• Archives of Pharmacal Research
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• v.28 no.7
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• Tuberculosis and Respiratory Diseases
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• v.47 no.2
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• pp.172-183
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• 1999

Background: Nitric oxide is a short-lived effector molecule derived from L-arginine by the nitric oxide synthase(NOS). Nitric oxide plays a role in a number of physiologic and pathophysiologic functions including host defense, edema formation, and regulation of smooth muscle tone. Some kinds of cells including macrophage are known to produce large quantities of nitric oxide in response to inflammatory stimuli such as interleukin-$1\beta$(IL-$1\beta$), tumor necrosis factor-$\alpha$(TNF-$\alpha$), interferon-$\gamma$(IFN-$\gamma$) and lipopolysaccharide(LPS). Reactive oxygen species are also known to be important in the pathogenesis of acute cell and tissue injury such as acute lung injury model Methods: Using the RA W264.7 cells, we have examined the ability of oxidant hydrogen peroxide($H_2O_2$) to stimulate nitric oxide production and inducible NOS mRNA expression. Also, we have examined the effects of NOS inhibitors and antioxidants on $H_2O_2$ induced nitric oxide production. Results: Stimulation of RAW264.7 cells with combinations of 100 ng/ml IL-$1\beta$, 100 ng/ml TNF-$\alpha$, and 100 U/ml IFN-$\gamma$ or 100 U/ml IFN-$\gamma$ and $1{\mu}g/ml$ LPS induced the synthesis of nitric oxide as measured by the oxidation products nitrite($NO_2^-$) and nitrate($NO_3^-$). Addition of $250 {\mu}M-2$ mM $H_2O_2$ to the cytokines significantly augmented the synthesis of $NO_2^-$ and $NO_3^-$(p<0.05). When cells were incubated with increasing concentrations of $H_2O_2$ in the presence of IL-$1\beta$, TNF-$\alpha$ and IFN-$\gamma$ at constant level, the synthesis of $NO_2^-$ and $NO_3^-$ was dose-dependently increased(p<0.05). $N^G$-nitro-L-arginine methyl ester(L-NAME), dose dependently, significantly inhibited the formation of $NO_2^-$ and $NO_3^-$ in cells stimulated with LPS, IFN-$\gamma$ and $H_2O_2$ at constant level(p<0.05). Catalase significantly inhibited the $H_2O_2$-induced augmentation of cytokine-induced $NO_2^-$ and $NO_3^-$ formation(p<0.05). But, boiled catalase did not produce a significant inhibition in comparison with the native enzyme. Another antioxidant 2-mercaptoethanol and orthophenanthroline dose-dependently suppressed $NO_2^-$ and $NO_3^-$ synthesis(p<0.05). Northern blotting demonstrated that H:02 synergistically stimulated the cytokine-induced iNOS mRNA expression in RA W264.7. Conclusion: These results suggest that $H_2O_2$ contributes to inflammatory process by augmenting the iNOS expression and nitric oxide synthesis induced by cytokines.

• Journal of Sericultural and Entomological Science
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• v.31 no.1
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• pp.37-44
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• 1989

The parasporal crystals of Bacillus thuringinsis subspecies kurstaki, dendrolimus, finitimus, aizawai and israelensis were compared by polyacrylamide electrophoresis, amino acid composition and immunological analysis. In the subspecies of kurstaki, dendrolimus, finitimus and aizawai, the molecular weights of main subusnits of crystal solubilized by alkaline solution were 1.3${\times}$105 and 6.5${\times}$104 while those of subsp. israelensis were 4${\times}$104 and 1,4${\times}$104. The degradation of lepidopteran toxic subspecies crystals by silkworm midgut protease showed 6.0-6.4${\times}$104 molecular weight and the pattern of degradation of subsp. israelensis crystals was similar to that of alkaline solution treatment. In the amino acid composition, aspartic acid in subsp. israelensis and glutiamic acid in the other four subspecies were the most abundant. The immunological characteristics of the crystals revealed that the antibody produced against the alkali-solubilized crystal protein of subsp. israelensis reacted with only its antigen, but the crystal antigens from the other four lepidopteran toxic subspecies did cross-react with each other as well as with their own homologous antisera.

• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.196-202
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• 2007

Mutations in the cystathionine ${\beta}$-synthase (CBS) gene cause homocystinuria, the most frequent inherited disorder in sulfur metabolism. CBS is the unique enzyme using both heme and pyridoxal 5-phosphate (PLP) for activity. Among the reported 140 mutations, one of the most common disease-causing alterations in human CBS is G307S mutation. To investigate the pathogenic mechanism of G307S by spectroscopic methods, we engineered the full length and the truncated G247S mutation of yeast CBS that is corresponding mutation to human G307S. Yeast CBS does not contain heme and thus gives a merit to study the spectroscopic properties. The UV-visible spectra of the purified full length and the truncated G247S yeast CBSs showed the total absence of PLP in the protein. The absence of PLP in G247S mutation was also confirmed by the PLP-cyanide adduct formation experiment, which was conducted by the incubation of the purified enzyme with KCN. The adducts were detected using a circular dichroism (CD) and a spectrofluorimeter. Radio isotope activity assay of full length and truncated G247S proteins also gave no activity. Our yeast G247S mutation data suggested that G307S might make the distortion of the active site so that cofactor PLP and substrate can not fit inside the active site. Our yeast CBS study addressed the reason why the G307S mutation in human CBS makes the enzyme inactive that consequently leads to severe clinical phenotype.

• Applied Biological Chemistry
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• v.20 no.1
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• pp.33-42
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• 1977

Asparagine biosynthesis by soybean sprouts grown under the dark conditions has been demonstrated. The amount of free asparagine synthesized in ten day-old soybean sprouts increases to 22.7% on the dry weight base. The effects of nitrogen compounds such as $NH_4Cl,\;(NH_4)_2SO_4$ and urea on asparagine synthesis during the sprouting were examined and the results showed that urea was more effective than other two compounds. Glutamine-dependent asparagine synthetase was partially purified (8.6 folds) through ammonium sulfate fractionation, followed by Sephadex G-150 gel filtration. The enzyme was very labile and required protection by thiol groups or high level of glycerol. The mixture of ATP and $Mg^{++}$ ion also stabilized the enzyme activity. The enzyme utilized glutamine more effectively than ${NH_4}^+$ as an amide donor for the formation of asparagine. The enzyme required L-aspartate (Km=3.1 mM), L-glutamine, ATP and $Mg^{++}$. It showed pH optimum of 7.5 and catalyzed the formation of ${\beta}-aspartyl$ hydroxamate in the presence of L-aspartate, ATP, $Mg^{++}$ and $NH_2OH$ in the reaction mixture.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.121-126
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• 2012

The objective of this study was to examine the effect of in vitro culture media on embryonic development of in vitro-matured (IVM) oocytes after parthenogenetic activation (PA) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 22~26 h. IVM oocytes were activated by electric pulses and cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 supplemented with essential and non-essential amino acids (NCSU-23aa). These media were further modified by supplementing 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and $10{\mu}M$ ${\beta}$-mercaptoethanol (designated as mPZM-3 and mNCSU-23aa, respectively). Culture of PA embryos in mPZM-3 significantly increased development to the blastocyst stage than culture in NCSU-23aa (36.2% vs. 24.8%, p<0.05). Modified PZM-3 showed a significantly higher blastocyst formation than NCSU-23aa in both groups of embryos that were activated at 44 h and 48 h of IVM (51.0% vs. 35.5% and 49.0% vs. 34.2% in oocytes activated at 44 h and 48 h of IVM, respectively). Irrespective of the follicle diameter where oocytes were collected, embryonic development to the blastocyst stage was increased (p<0.05) by the culture in mPZM-3 compared to culture in NCSU-23aa (25.9% vs. 34.2% and 32.9% vs. 44.8% in embryos derived from small and medium size follicles, respectively). Our results demonstrated that culture media had significant effect on preimplantation development PA embryos and that mPZM-3 was superior to mNCSU-23 in supporting development to the blastocyst stage in pigs. This beneficial effect of mPZM-3 on embryonic development was not impaired by other factors such as time of oocyte activation and origin of immature oocytes (small and medium size follicles).

• Journal of Microbiology and Biotechnology
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• v.27 no.12
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• pp.2190-2198
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• 2017

Thermoactinomyces sp. strain YT06 was isolated from poultry compost and observed to degrade integral chicken feathers completely at $60^{\circ}C$, resulting in the formation of 3.24 mg/ml of free amino acids from 50 ml of culture containing 10 g/l chicken feathers. Strain YT06 could grow and secrete keratinase using feather as the only carbon and nitrogen sources without other supplement, but complementation of 10 g/l sucrose and 4 g/l $NaNO_3$ increased the production of the keratinolytic enzyme. The maximum protease activity obtained was 110 U/ml and for keratinase was 42 U/ml. The keratinase maintained active status over a broad pH (pH 8-11) and temperature ($60-75^{\circ}C$). It was inhibited by serine protease inhibitors and most metal ions; however, it could be stimulated by $Mn^{2+}$ and the surfactant Tween-20. A reductive agent (${\beta}$-mercaptoethanol) was observed to cleave the disulfide bond of keratin and improve the access of the enzyme to the keratinaceous substrate. Zymogram analysis showed that strain YT06 primarily secreted keratinase with a molecular mass of approximately 35 kDa. The active band was assessed by MALDI-TOF mass spectrometry and was observed to be completely identical to an alkaline serine protease from Thermoactinomyces sp. Gus2-1. Thermoactinomyces sp. strain YT06 shows great potential as a novel candidate in enzymatic processing of hard-to-degrade proteins into high-value products, such as keratinous wastes.

• BMB Reports
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• v.35 no.5
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• pp.524-531
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• 2002

Thrombotic thrombocytopenic purpura (TTP) is characterized by widespread platelet thrombi in arterioles and capillaries. Unusually large or multimeric von Willebrand factor, as well as one or ore platelet-agglutinating factors, have been implicated in the pathogenesis of TTP. But, the actual mechanisms of platelet agglutination have not been satisfactorily explained. Recent studies suggested the 37-kDa platelet-agglutinating protein (PAP) p37 to be partially responsible for the formation of platelet thrombi in patients with TTP. We studied mobility in SDS-PAGE, the sequence of N-terminal amino acid residues, DNA and antigenic characteristics of PAP p37, which might be related to the pathogenesis of TTP. PAP p37 was purified from the plasma of a 31-year-old male Korean patient with acute TTP. The findings are as follows: (1) We compared PAP p37 with thrombin through the use of SDS-PAGE, either with or without $\beta$-mercaptoethanol. PAP p37 did not appear to be cleaved between the A- and B-chains of prethrombin 2. However, thrombin did cleave between those of prethrombin 2, but linked with disulfide bridge. (2) N-terminal 21 amino acid sequence of PAP p37 was T-F-G-S-G-E-A-D-X-G-L-R-P-L-F-E-K-K-S-L-E. It appeared to be identical to that of 285-305 amino acid residues of human prothrombin (prethrombin 2). (3) No prothrombin gene DNA mutation was revealed. (4). The antigenicity of PAP p37 was similar to thrombin, which was a result of the competitive binding against the anti-thrombin antibody. With these results, we conclude that PAP p37 has similar characteristics to prethrombin2.

• Journal of Microbiology and Biotechnology
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• v.18 no.9
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• pp.1555-1563
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• 2008

A novel $\alpha$-amylase ($\alpha$-1,4-$\alpha$-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal a-amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and $55^{\circ}C$ with an apparent $K_m$ value of 1.66 mg/ml and $V_{max}$ of 0.1${\mu}mol$glucose $min^{-1}$ $ml^{-1}$. ScAmy43 activity was strongly inhibited by $Cu^{2+}$, $Mn^{2+}$, and $Ba^{2+}$, moderately by $Fe^{2+}$, and was only weakly affected by $Ca^{2+}$ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and $\beta$-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.