• The Korean Journal of Physiology and Pharmacology
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• v.17 no.1
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• pp.51-56
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• 2013

Many intracellular proteins and signaling cascades contribute to the sensitivity of N-methyl-D-aspartate receptors (NMDARs). One such putative contributor is the serine/threonine kinase, protein kinase C (PKC). Activation of PKC by phorbol 12-myristate 13-acetate (PMA) causes activation of extracellular signal-regulated kinase (ERK) and promotes the formation of new spines in cultured hippocampal neurons. The purpose of this study was to examine which PKC isoforms are responsible for the PMA-induced augmentation of long-term potentiation (LTP) in the CA1 stratum radiatum of the hippocampus in vitro and verify that this facilitation requires NMDAR activation. We found that PMA enhanced the induction of LTP by a single episode of theta-burst stimulation in a concentration-dependent manner without affecting to magnitude of baseline field excitatory postsynaptic potentials. Facilitation of LTP by PMA (200 nM) was blocked by the nonspecific PKC inhibitor, Ro 31-8220 ($10{\mu}M$); the selective $PKC{\delta}$ inhibitor, rottlerin ($1{\mu}M$); and the $PKC{\varepsilon}$ inhibitor, TAT-${\varepsilon}V1$-2 peptide (500 nM). Moreover, the NMDAR blocker DL-APV ($50{\mu}M$) prevented enhancement of LTP by PMA. Our results suggest that PMA contributes to synaptic plasticity in the nervous system via activation of $PKC{\delta}$ and/or $PKC{\varepsilon}$, and confirm that NMDAR activity is required for this effect.

• Journal of Mushroom
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• v.3 no.2
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• pp.90-94
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• 2005

This study was carried out to investigate the biological activities of P. tenuipes formed on silkworm (Bombyx mori) variety. The mean content of total amino acid in the fruiting bodies of P. tenuipes was $1.03{\mu}mole/g$. The distribution rate of amino acid components decreased in the order of Arginine(12.2%) > Glycine(10.5%) > Proline(9.6) > Tyrosine(8.9%) > Serine > Leucine > Threonine. The most abundant amino acid in the fruiting bodies of the Baegokjam, Chilbojam and Hachojam infected with P. tenuipes was arginine, while Yangwonjam was Glycine. The most abundant fatty acid in P. tenuipes was Oleic acid on a dry weight basis. The unsaturated fatty acids such as Oleic acid, Linoleic acid and Linolenic acid accounted for more than 78% of the total fatty acids.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.281-286
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• 2010

This study was to evaluate the protein profile of seminal plasma using 2-DE in Hanwoo. Seminal plasma was harvested from five mature Hanwoo, and seminal plasma protein was extracted by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was $300\;{\mu}l$. Immobilized pH gradient (IPG) strip was used 18 cm and 3~11 NL. SDS-PAGE was used 12% acrylamide gel. Each gels were visualized by comassie brilliant blue and silver staining. These spots were analyzed by MALDI-TOF MS and searched on NCBInr. The result, 20 proteins of 36 protein spots were searched through peptide sequencing on the NCBInr. 8 proteins profiled by 2-DE were proved through previous bovine studies and the name of each protein was albumin, nucleobindin, clusterin, TIMP-2, spermadhesin Z13, spermadhesin-1 and BSP proteins (BSP 30 kDa and BSP A1/A2). 12 new proteins were ATP synthase, protein MAK16 homolog, Transmembrane protein 214, E3 ubiquitin-protein ligase BRE1A, dual serine/threonine and tyrosine protein kinase, tissue factor pathway inhibitor 2, alpha-actinin-4, RUN domain-containing protein 3B, catenin alpha-1, protein-glutamine gamma-glutamyltransferase 2, plakophilin-1 and inter-alpha-trypsin inhibitor heavy chain H1 has not been previously described in the bovine seminal plasma study. These proteins may be contribute to define the type of proteins affecting fertility of male and improve the fertilizing ability of semen in Hanwoo.

• Journal of Life Science
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• v.14 no.2
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• pp.252-254
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• 2004

The mutant tryptophan synthases at $\alpha$Asp$^{56}$ have low catalytic activities. The enzymes were treated with $\alpha$ and $\beta$ subunit-specific ligands in the presence of L-serine and indoline. It was shown that the cations resulted in changes of absorbance patterns among the proteins, while glycerophosphate showed similar pattern of absorbance. The glycerophosphate binds to the active site of $\alpha$ subunit so that $\alpha$Asp$^{56}$ may not be involved in the allosteric control with the active site of $\alpha$ subunit occupied by substrate. The results suggest that $\alpha$Asp$^{56}$ may playa role in the step of a series of reaction occurring without bound substrates in the active site of asubunit.

• Tuberculosis and Respiratory Diseases
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• v.72 no.4
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• pp.343-351
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• 2012

Background: The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit this pathway are currently under development for lung cancer treatment. In the present study, we have tested whether dual inhibition of PI3K/Akt/mTOR signaling can lead to enahnced antitumor effects. We have also examined the role of autophagy during this process. Methods: We analyzed the combination effect of the mTOR inhibitor, temsirolimus, and the Akt inhibitor, GSK690693, on the survival of NCI-H460 and A549 non-small cell lung cancer cells. Cell proliferation was determined by MTT assay and apoptosis induction was evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Autophagy induction was also evaluated by acridine orange staining. Changes of apoptosis or autophagy-related proteins were evaluated by western blot analysis. Results: Combination treatment with temsirolimus and GSK690693 caused synergistically increased cell death in NCI-H460 and A549 cells. This was attributable to increased induction of apoptosis. Caspase 3 activation and poly(ADP-ribose) polymerase cleavage accompanied these findings. Autophagy also increased and inhibition of autophagy resulted in increased cell death, suggesting its cytoprotective role during this process. Conclusion: Taken together, our results suggest that the combination of temsirolimus and GSK690693 could be a novel strategy for lung cancer therapy. Inhibition of autophagy could also be a promising method of enhancing the combination effect of these drugs.

• Korean Journal of Veterinary Research
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• v.39 no.3
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• pp.545-553
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• 1999

A 474-base pair segment covering the hypervariable region of VP2 gene from six Korean infectious bursal disease virus(K-IBDV) isolates(K1, K2, SH/92, 225, 269, 310) and one attenuated IBDV(DAE) were amplified using RT-PCR, sequenced, and compared with published sequences for IBDV. K-IBDV isolates(K1, K2, SH/92, 225, 269) and foreign very virulent(vv) IBDV strains had 94.93~100% amino acid sequence similarity. K-IBDV isolate 310 and other K-IBDV isolates had 84.31~86.07% amino acid sequence similarity. Attenuated strain(DAE), like other attenuated strain, has substitution at positions 279(D to N) and 284(A to T) as well as in the serine-rich heptapeptide region. Five K-IBDV isolates except 310 isolate share unique amino acid residues at positions 222(A), 256(I), 294(I) which are not present in other standard and attenuated strains. At the two hydrophilic region, K-IBDV isolates except 310 isolate had identical amino acids comparing with Belgium vv IBDV 894VB but had four amino acid substitutions comparing with Chinese vv IBDV F9502. The SWSASGS heptapeptide is conserved in all K-IBDV isolates. The sequence of K-IBDV isolate 310 was markedly different from other IBDV strains, evolving from a separate lineage than the others. By phylogenetic analysis, Five K-IBDV isolates except 310 isolate were categorized in one group with foreign vv IBDV isolates but K-IBDV isolate 310 was categorized in a separate group which was differentiated from other compared IBDV strains.

• BMB Reports
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• v.37 no.4
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• pp.429-438
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• 2004

Complementary DNAs encoding $\alpha$-amylases (Amyl I, Amyl III) and glucoamylase (GA I) were cloned from Aspergillus awamori KT-11 and their nucleotide sequences were determined. The sequence of Amyl III that was a raw starch digesting $\alpha$-amylase was found to consist of a 1,902 bp open reading frame encoding 634 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. On the other hand, the sequence of Amyl I, which cannot act on raw starch, consisted of a 1,500 bp ORF encoding 499 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. The sequence of GA I consisted of a 1,920 bp ORF that encoded 639 amino acids. The signal peptide was composed of 24 amino acids. The amino acid sequence of Amyl III from the N-terminus to the amino acid number 499 showed 63.3% homology with Amyl I. However, the amino acid sequence from the amino acid number 501 to C-terminus, including the raw-starch-affinity site and the TS region rich in threonine and serine, showed 66.9% homology with GA I.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.798-803
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• 1999

Prolyl endopeptidase [PEP; EC 3.4.21.26], a serine protease which is known to cleave peptide bonds on the carboxy side of a proline residue, plays an important role in the degradation of proline-containing neuropeptides that have been suggested to participate in learning and memory processes. An abnormal increase in the level of PEP, which can lead to generation of $A{\beta}$, is also suggested to be involved in Alzheimer's type senile dementia. In the course of screening PEP inhibitors from Basidiomycetes, the mushroom Polyozellus multiplex exhibited a high inhibitory activity against PEP. Two active compounds were isolated from the ethyl acetate soluble fraction by consecutive purification, using silica gel, Sephadex LH-20, and Lobar RP-18 chromatography. The chemical structures of these compounds were identified as thelephoric acid and 12-acety1-2,3,7,8-tetrahydroxy-[12H]-12-hydroxymethylbenzobis[I.2b,3.4b'] benzofuran-11-one (kynapcin-9) by spectral data including UV, IR, MS, HR-MS, $^1H-,{\;}^{13}C-$, and 2D-NMR. The $IC_{50}$ values of the thelephoric acid and kynapcin-9 were 0.157 ppm (446nM) and 0.087 ppm (212nM) and their inhibitor constants ($K_i$) were 0.73ppm ($2.09{\;}\mu\textrm{m}$) and 0.060 ppm (146 nM), respectively. Furthermore, they were non-competitive with a substrate in Dixon plots.

• Archives of Pharmacal Research
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• v.25 no.4
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• pp.505-510
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• 2002

An improved kinetic assay for prekallikrein activator (PKA), a potential vasodilator, has been developed to be used as an indicator for quality control during production of human albumin preparations. It consists of two reaction stages. In the first stage, PKA and prekallikrein are incubated at $37^{\circ}C$ for 45 min to allow the transformation into kallikrein. Kallikrein, a serine protease, catalyzes the splitting of p-nitroaniline (pNA) from its substrate H-D-Pro-Phe-Arg-pNA(S-2302). The rate at which pNA is released was measured spectrophotometrically at 405 nm. Prekallikrein, a substrate of PKA was purified by DEAE ion-exchange chromatography and the major potential variations in the assay were optimized; pH 8.0 and 150 mM sodium chloride were chosen to give a proper ionic strength. Reaction times in the range of 10 to 360 min provided linear dose-response curves. The concentration of prekallikrein was adjusted to fall between 1:1 and 1:3 dilutions to generate a linear standard calibration curve. Under the optimized conditions, reproducibility was checked. In a precision test, the coefficient of variation (CV) stayed within ${\pm}4%$ and the dose-response curve showed a good correlation (${r^2}=0.999$). An accuracy test with an international standard of PKA afforded a mean recovery of 97.5%.

• Journal of Microbiology and Biotechnology
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• v.34 no.2
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• pp.436-456
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• 2024

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca²⁺ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.