• Journal of Korean Society of Forest Science
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• v.81 no.3
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• pp.273-279
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• 1992

Protoplasts isolated from leaf mesophyll tissues of Populus koreana ${\times}$ P. nigra var. italica were fused with those of P. euramericana cv. Guardi. Well expended healthy leaves of 5 to 7 week-old-plantlet grown in vitro were used as source materials. Leaves from P. koreana ${\times}$ P. nigra var. italica and P. euramericana cv. Guardi were digested in enzyme solution I (2.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v) and enzyme solution II (1.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v), respectively, The highest frequency of fusion among the protoplasts originated from the two source materials was approximately 21% using 40% PEG or 15% dextran. In addition, fusion frequency was enhanced by incorporating 30mM of $Ca^{2+}$ in eluting solution at pH 10.5. Dividing cells and/or mint-calli were obtained by culturing the fusion products in a liquid 8p-KM medium supplemented with 0.6M sucrose, $0.45{\mu}M$ 2, 4-D, and $0.5{\mu}M$ BA. Shoots were regenerated from the fusion product-derived calli after culture on MS medium containing $5.0{\mu}M$ zeatin. To verify the putative hybrid or cybrid, SDS-PAGE was carried out. From the 24 regenerants, just two plants showed intermediate protein band patterns compared with those of the original source plants.

• 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.

• Journal of Life Science
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• v.23 no.8
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• pp.1050-1056
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• 2013

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia throughout the world. The bacteria invade through lung tissue and cause sepsis, shock, and serious sequelae, including rheumatic fever and acute glomerulonephritis. However, the molecular mechanism associated with pneumonia's penetration of lung tissue and invasion of the blood stream are still unclear. We attempted to investigate the host cell response at protein levels to S. pneumoniae D39 invasion using human lung cancer epithelial cells, A549. Streptococcus pneumoniae D39 began to change the morphology of A549 cells to become round with filopodia at 2 hours post-infection. A549 cell proteins obtained at each infection time point were separated by SDS-PAGE and analyzed using MALDI-TOF. We identified several endoplasmic reticulum (ER) resident proteins such as Grp94 and Grp78 and mitochondrial proteins such as ATP synthase and Hsp60 that increased after S. pneumoniae D39 infection. Cytosolic Hsc70 and Hsp90 were, however, identified to decrease. These proteins were also confirmed by Western blot analysis. The identified ER resident proteins were known to be induced during ER stress signaling. These/ data, therefore, suggest that S. pneumoniae D39 infection may induce ER stress.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.5
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• pp.637-644
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• 1998

[ $\beta$ ]-Galactosidase was extracted from the internal organ of sea urchin, Hemicentrotus pulcherrimus The enzyme was purified 384.6-fold over the crude extract by the sequential chromatographic methods including DEAE-Sephadex A-25, CM-Cellulose, and Con A-Sepharose 4B affinity chromatography with a recovery $1.26\%$. The molecular weight of the purified enzyme was estimated approximately 94 kDa as monomeric term by SDS-PAGE and Sephadex G-150 gel chromatography. The maximum enzymatic activity was observed at pH 3.0 and $50^{\circ}C$ but the one was stable over the ph range or 3.0$\~$5.0 and below $37^{\circ}C$. The $K_m$ and $V_{max}$ values against PNPG (P-nitrophenyl $\beta$-D-galactopyranoside) were 15.0 mM and 214 $\mu$mole/min per mg protein, respectively. The enzymatic activity was activated by $Ba^{2+}$, but significantly inhibited by $DEP,\;Hg^{2+},\;Sn^{2+}$ and galactose.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1057-1062
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• 2004

The entomopathogenic bacterium Bacillus thuringiensis is the most widely used biopesticide. Insecticidal proteins, coded by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. We isolated a Bacillus thuringiensis strain having insecticidal activity against larvae of the house fly (M. domestica) from the soils at a pig farm in Korea, and named it Bacillus thuringiensis SM. The culture filtrate from Bacillus thuringiensis SM showed strong lethality (83.3%) against M. domestica larvae. The parasporal crystal is enclosed within the spores' outermost envelope, as determined by transmission electron microscopy, and exhibited a bipyramidal form. The crystal proteins of strain SM consisted of five proteins with molecular weights of approximately ~130, ~80, ~68, ~42, and ~27 kDa on a 10% SDS-PAGE (major band, a size characteristic of Cry protein). Examination of antibiotic resistance revealed that the strain SM showed multiple resistant. The strain SM had at least three different plasmids with sizes of 6.6, 9.3, and 54 kb. Polymerase chain reactions (PCRs) revealed the presence of cry1, cry4A2, and cry11A1 genes in the strain SM. The cry1 gene profile of the strain SM appeared in the three respective products of 487 bp [cry1A(c)], 414 bp [cry1D], and 238 bp [cry1A(b)]. However, the strain SM has not shown the cry4A2 md cry11A1 genes. In in vivo toxicity assays, the strain SM showed high toxicity on fly larvae (M. domestic) [with $LC_{50}$ of 4.2 mg/ml, $LC_{90}$ of 8.2 mg/ml].

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.107-113
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• 1997

A strain producing a high amount of chitinase was isolated from soil, identified as Pseudomonas sp., and tentatively named Pseudomonas sp. YHS-A2. An extracellular chitinase of Pseudomonas sp. YHS-A2 was purified according to the procedure of ammonium sulfate saturation, affinity adsorption, Sephadex G-100 gel filtration and Phenyl-sepharose CL-4B hydrophobic interaction column chromatography. The molecular weight of the purified enzyme was estimated to be 55 kDa on SDS-PAGE was confirmed by active staining. Optimal pH and temperature of the enzyme are pH 7.0 and $50^{\circ}C$, respectively, and the enzyme is stable between pH 5.0 and 8.0 and below $50^{\circ}C$. The main products of colloidal chitin by the chitinase were N-acetyl-D-glucosamine and N,N'-diacetylchitobiose both of which were detected by HPLC analysis. The enzyme is supposed to be a random-type endochitinase which can degrade any position of ${\beta}$-l,4-linkages of chitin and chitooligosaccharides. The chitinase inhibited the growth of some phytopathogenic fungi, Fusarium oxysporum, Botrytis cineria, and Mucor rouxii and these antifungal effects were thought to be due to the characteristics of endochitinase.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.541-546
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• 1997

There are two conserved regions with a significantly high amino acid sequence homology among the A subunits of STX, SLTs and ricin. To produce an inactive Verotoxin-2 (VT-2), two different mutants, pE167D and pDE5A, were constructed by site-directed mutagenesis, respectively, on the basis of the previous reports that two regions lie within the active-site clefts of the A subunits of ricin and STX family. The cytotoxicity ($10^3$ $CD_{50}/ml$) of VT-2 holotoxin with E167D mutation was reduced by $10^3$-fold compared with wild-type level. In addition, VT-2 with DE5A ($Trp_{202}GlyArgIleSer_{206}$) deletion mutation showed a significantly low cytotoxicity ($10^1$ $CD_{50}/ml$), resulting in $10^5$- and $10^2$-fold reductions, respectively, compared with the wild-type and E167D mutatant. SDS-PAGE for protein samples showed a 33-kDa band corresponding to the A subunit of VT-2. These results indicate that reduction in cytotoxic activity was affected not by amount of VT-2 protein produced but by mutation.

• Journal of Veterinary Clinics
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• v.25 no.3
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• pp.152-158
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• 2008

We investigated the changes of protein in chronic MI which was occurred with long-term ischemia, without reperfusion. Sprague Dawley (SD) rats were divided into the sham group and the experimental groups (MI groups). The sham group was treated only thoracotomy without ligation for left main descending artery (LMDA) of left coronary artery (LCA), and the experimental groups (MI7d, ligation of LMDA for 7 days and MI30d, ligation of LMDA for 30 days) were conducted an artificial chronic MI. The change of proteins according to passage of times was compared and analyzed on first and second dimension (1 and 2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Among total 46 spots expressed differentially in the sham group versus MI7d and MI30d groups on 2D gel, we selected proteins that the volume of spot was increased in the MI7d and MI30d groups compared with the sham group. After that, the proteins were identified through liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. In result, we could obtain many proteins as follows; albumin, glucose regulated protein 58 KDa, similar to tripartite motif protein 50, ubiquinol-cytochrome c reductase core protein II, sarcomeric mitochondrial creatine kinase, ATP synthetase alpha chain (mitochondrial precursor) and creatine kinase. In conclusion, we suggest many changed proteins shown at chronic ischemia after artificial MI and consider that these proteins play an important role in the function of heart after MI.

• The Korean Journal of Pharmacology
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• v.31 no.2
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• pp.207-217
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• 1995

To investigate the functional and immunological properties of the Ca-release channel in the sarcoplasmic reticulum(SR) of the eel skeletal muscle, $[^3H]ryanodine$ binding, SDS gel electrophoresis, $^{45}Ca\;release$ studies, and immunoblot assay were carried out in the SR of the eel skeletal muscle. Maximal binding sites(Bmax) and $K_D$ values of $[^3H]ryanodine$ for Ca-release channel of the SR of the eel skeletal muscle were $19.44{\pm}1.40\;pmole/mg$ protein and $15.55{\pm}1.69\;nM$, respectively. $[^3H]Ryanodine$ binding to RyR was increased by calcium and AMP. The SR of the eel skeletal muscle has two high molecular weight bands on the SDS PAGE. The mobility of upper band was more slower than the single band of the rabbit skeletal muscle, and that of the lower band was similar with the single band of canine cardiac muscle. Vesicular $^{45}Ca-release$ was activated by calcium. Ca-induced $^{45}Ca-release$ was significantly inhibited by $MgCl_2(2\;mM)$, ruthenium red$(10\;{/mu}M)$ or tetracaine(1 mM), but not by high concentration of calcium itself. AMP-induced $^{45}Ca-release$ was slightly occurred only in the absence of calcium, it was not inhibited by $MgCl_2$ or ruthenium red. Caffeine also increased $^{45}Ca-release$ from the SR vesicles, but it was not affected by $MgCl_2$ or ruthenium red. Polyclonal Ab against rat skeletal muscle RyR is reacted with that of rabbit, but not reacted with that of the eel skeletal muscle. These results suggested that ryanodine receptor of the SR of the eel skeletal muscle is showing some similar properties with that of mammalian skeletal muscle, but might be an another isotype channel having two bands which is less sensitive to AMP, not cross-reacted with antisera against rat RyR, and not inhibited by high concentration of calcium.

• Journal of Microbiology and Biotechnology
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• v.21 no.6
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• pp.574-581
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• 2011

A Gram-positive bacterium was isolated from the saline soils of Jangpura (U.P.), India, and showed high-level of radiation-resistant property and survived upto 12.5 kGy dose of gamma radiation. The 16S rDNA sequence of this strain was examined, identified as Bacillus sp. strain HKG 112, and was submitted to the NCBI GenBank (Accession No. GQ925432). The mechanism of radiation resistance and gene level expression were examined by proteomic analysis of whole-cell extract. Two proteins, 38 kDa and 86.5 kDa excised from SDS-PAGE, which showed more significant changes after radiation exposure, were identified by MALDI-TOF as being flagellin and S-layer protein, respectively. Twenty selected 2-DE protein spots from the crude extracts of Bacillus sp. HKG 112, excised from 2- DE, were identified by liquid chromatography mass spectrometry (LC-MS) out of which 16 spots showed significant changes after radiation exposure and might be responsible for the radiation resistance property. Our results suggest that the different responses of some genes under radiation for the expression of radiation-dependent proteins could contribute to a physiological advantage and would be a significant initial step towards a fullsystem understanding of the radiation stress protection mechanisms of bacteria in different environments.