• Biomedical Science Letters
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• v.16 no.4
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• pp.259-264
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• 2010

This study was designed to investigate the effects of nitric oxide on the neuronal activity of rat cerebellar Purkinje cells. Sprague-Dawley rats aged 14 to 16 days were decapitated under ether anesthesia. After treatment with pronase and thermolysin, the dissociated Purkinje cells were transferred into a chamber on an inverted microscope. Spontaneous action potentials and potassium current were recorded by standard patch-clamp techniques under current and voltage-clamp modes respectively. 15 Purkinje cells revealed excitatory responses to $20\;{\mu}M$ of sodium nitroprusside (SNP) and 4 neurons (20%) did not respond to SNP. Whole potassium currents of Purkinje cells were decreased by SNP (n=10). Whole potassium currents of Purkinje cells were also decreased by L-arginine, substrate of nitric oxide (n=10). These experimental results suggest that nitric oxide increases the neuronal activity of Purkinje cells by altering the resting membrane potential and after hyperpolarization.

• Proceedings of the Ginseng society Conference
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• 1978.09a
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• pp.75-77
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• 1978

It was found that water extract of Panax ginseng strongly inhibited adrenaline-induced lipolysis in isolated fat cells of rat epididymal adipose tissue. An antilipolytic action of the water extract was easily inactivated by treatment with pronase, suggesting that the active principle might be a protein or a peptide. Experiments were designed to purify the antilipolytic substance, or insulin-like substance, of the water extract. The water extract was dialyzed against disti'led water. The outer dialysate was subjected to DEAE-cellulose column chromatography, gelfuaration on sephadex G-50 column, avicel cellulose column chromatography and phospho-cellulose column chromatography, successively. The finally purified substance gave one spot on thin layer chromatography. The molecular weight was found to be around 1000. Experiments are now in progress to elucidate the structure of this insulin-like peptide.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.111-116
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• 1994

This study was carried out to investigate the aggregation rate of isolated mouse 2-, 4- and 8-cell stage blastomeres in phytohemagglutinin(PHA) solution. The morphologically normal embryos were collected from the oviduct of superovulated female mouse by flushing with M2 and the zona pellucida of embryos were removed with 0.5% pronase. The blastomeres were isolated by pipetting after plunging into Ca++-Mg++free PBS for 20 min. The result showed that aggregation rate in 0.5% (84.9~93.1%) was higher than that in 1.0% PHA(76.0~82.1%). Optimal aggregation time was 60min (83.9~100.0%) when compared with 30min (78.8~87.5%). Developmental to blastocyst in recombinated blastomeres was higher under conditions of 0.5% PHA solution and 60-min aggregation than that under other conditions.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.249-254
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• 1995

When mating type A and a cells of heterobasidiomycetous yeast Rhodosporidium toruloides were mix-cultured, both of the mating type cells have shown strong agglutination. But this agglutination was not detactable when the A and a cell were cultured separately. From reagglutination made just after the result of disassembling the agglutination by sonication, we knew that the agglutination was sexual-agglutination, not simple physical cell agglutination. The sexual agglutination was progressed actively on logarithmic phase and, in addition, progressed faster on mating type a cell treated with rhodotorucine A. These sexual agglutination have been inhibited by several protease such as trypsin, pronase, chymotrpysin and thermolysin and inhibited by 5 mM DTT as well.

• Korean Journal of Food Science and Technology
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• v.29 no.1
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• pp.1-8
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• 1997

A complement system-activating (anti-complementary) polysaccharide was purified from the hot water extract of young stems of Cinnamomum cassia Blume. Crude polysaccharide fraction (CC-1) was prepared from the hot water extract of the young stems followed by methanol-reflux, precipitation with ethanol, dialysis, and lyophilization. The anti-complementary activity of CC-1 was decreased greatly by periodate oxidation, but was not changed by pronase digestion. These suggest that carbohydrate moiety may be related to the activation of complement system. According to its ionic strength CC-1 was fractionated first using cetavlon to give 4 fractions, CC-2, 3, 4 and 5. Among them CC-2 fraction was found to retain the highest activity and yield. CC-2 was separated to an unabsorbed neutral sugar portion (CC-2-I) and seven absorbed acidic sugar fractions $(CC-2-II{\rightarrow}CC-2-VIII)$ on DEAE-Toyopearl 650C (Cl-). CC-2-III showing higher anti-complementary activity and yield than those of other fractions, was further purified on the gel permeation of Sephadex G-100 and Sepharose CL-6B to CC-2-IIIa-3. CC-2-IIIa-3 was determined to have a homogeneity hy GPC (Sepharose CL-6B) and HPLC. Gel chromatography using standard dextrans gave a value of $2.4{\times}10^5$ for the molecular weight. The purified polysaccharide, CC-2-IIIa-3 consisted of arabinose, xylose, glucose, galactose, galacturonic acid and glucuronic acid in a molar ratio of 5.56 : 3.77 : 1.87 : 1.00 : 5.12 : 3.13 and contained no nitrogen.

• Applied Biological Chemistry
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• v.46 no.1
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• pp.60-65
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• 2003

MMP-1 inhibitory compounds were isolated from 120 Korean traditional edible plants. UP- 1 activity significantly increased linearly with increasing UVB dose in normal human foreskin fibroblast HS68 cell, showing maximum activity at approximately 35 $mJ/cm^2$, whereas in HaCaT cell, normal human keratinocyte, no increase was observed. Maximum secretion of MMP-1 after UVB treatment occurred around 36-48 k after treatment. MMP-1 inhibitory compound isolated from cold-water fraction of Cataegus pinnatifida Bunge showed the mort potent activity. The MMP-1 inhibitory compound was deduced as a peptide based on the fact that pronase digestion decreased the activity whereas periodate oxidation did not. The most potent UP- 1-inhibitory protein, CP-2Va-2, showing an activity of 88.5% against MMP-1, was isolated through sequential column chromatography on DEAE-Toyopearl 650C, Butyl-Toyopearl 650M, and Bio-Gel P-30. Molecular weight of CP-2Va-2 determined through high performance liquid chromatography and SDS PACE was 19 and 20 kDa. respectively, signifying a monomeric structure.

• Applied Biological Chemistry
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• v.41 no.1
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• pp.60-66
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• 1998

During the screening for anti-complementary activity from 10 kinds of edible mushrooms, an alkali extract of Agaricus bisporus showed the highest activity through the complement fixation test. The crude anti-complementary material(AB-0) from Agaricus bisporus was obtained by the alkali extraction using 1 N NaOH containing 5% urea$(65^{\circ}C)$, followed by methanol reflux, dialysis and lyophilization. The fraction AB-O showed potent anit-complementary and anti-tumor activity against sarcoma-180 injected mice. The fraction AB-0 was divided into 5 fractions(AB-20, AB-40, AB-60, AB-80, AB-A) by gradual acetone precipitation. Among them fraction AB-20 having the highest activity and yield was found to contain 39% carbohydrate and 46% protein. The anti-complementary protein-bound polysaccharide AB-20 consisted of glucose, arabinose, xylose, galactose and mannose in a molar ratio of 6.49 : 1.98 : 1.24 : 1.00 : 0.71, respectively and its main component amino acids were alanine(20.59%), isoleucine(16.85%), glutamine+glutamic acid(14.12%) and leucine (13.83%). The anti-complementary activity of AB-20 was decreased greatly by periodate oxidation, but decreased slightly by pronase digestion. This indicates that polysaccharide moiety is corelated with the anti-complementary activity and that protein is also involved in the activity.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.105-113
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• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• Applied Biological Chemistry
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• v.35 no.4
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• pp.308-314
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• 1992

To examine the characteristics of anti-complementary compounds from Arecae Pericarpium (the pericarps of Areca catechu) which showed the highest activity during our screening procedures, the extraction and purification were performed. AC-1 fraction from Arecae Pericarpium was purified by hot water extraction, methanol reflux, ethanol precipitation, dialysis and lyophilization. This compound had total sugar 48.2%, uronic acid 14.6% and protein 36.8%. Rhamnose, arabinose, mannose and galactose were found in sugar components. By cetavlon (cetyltrimethylammonium bromide) treatment AC-1 was fractionated to AC-2, AC-3 and AC-4. Among them, AC-2 showed the highest activity and yield. By periodate oxidation, AC-2 was deactivated, but had no change in activity by pronase digestion. Moreover active fractions, AC-2-IIIa and AC-2-IIIc isolated from AC-2 by two successive column chromatography using DEAE-Toyopearl $650C(Cl^-form)$ and Sephadex G-100. AC-2-IIIa was mainly made up of rhamnose, mannose, galactose and glucose, and AC-2-IIIc, mannose, galactose and glucose. These both polysaccharides were identified as homogeneous by gel filtration of Sepharose CL-4B and electrophoresis, and molecular weights of them were 120,000 and 15,000, respectively.

• Applied Biological Chemistry
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• v.13 no.1
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• pp.65-72
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• 1970

Two peptides (Im pr-M, Im ch-M) derived from Im ${\lambda}-type$ of Bence Jones Protein and one peptide (Ikch-M) from Ik were separated and purified using the Dowex $50{\times}2$ column $(1{\times}20\;cm)$ and Dowex $1{\times}2(0.9{\times}50\;cm)$. The buffer solution was composed of 1% pyridine and IM formic acid in Dowex $1{\times}2$ column. The blocked N-terminal was examined with ninhydrin reaction before and after alkaline hydrolysis, which was fractionated by Dowex $1{\times}2$ column. Pyrro-glutamic acid in N-terminal residue was identified by comparing with the authentic pyrro-glutamic acid through a high voltage electrophoresis (pH 3.5, 3000 V.) after the peptide Im pr-M (PCA. Ser) was cleavaged at the position of serine with cone. (12 N) HCl and the pyrro-glutamic acid was converted to glutamic acid by treating it with N-NaOH for 116 hours at $27^{\circ}C$. The substractive method was applied to find out the sequence of peptides and carboxypeptidase A was employed to release C-terminal residue from the peptide. In present study PCA. Ser in Im Pr-M was isolated from the pronase digested ${\lambda}$-type Bence Jones protein. The yield of the Im Pr-M was 79.6 percent of its theoretical value, based on the molecular weight of Bence Jones Protein. Im ch-M (PCA. Ser Val. Leu) was isolated from the chymotrypsin digested ${\lambda}$-type Bence Jones Protein. The yield of the Im ch-M was 72.2 percent. based on the molecular weight of Bence Jones Protein. Ik ch-M (PCA. Ser. Ala. Leu) was isolated from the chymotrypsin digested ${\lambda}$-type Bence Jones Protein and its yield was 42% based on the molecular weight of Bence Jones Protein.