• Journal of Life Science
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• v.22 no.2
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• pp.209-219
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• 2012

The properties of lactate dehydrogenase (LDH, EC 1.1.1.27) eye-specific $C_4$ isozyme were studied by polyacrylamide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme kinetics. Furthermore, we proposed the optimal conditions for measuring the activity of LDH eye-specific $C_4$ isozyme. The isozymes were detected in the cytosol of eye tissues from Lepomis macrochirus and Micropterus salmoides and were more similar to the $A_4$ than the $B_4$ isozyme. LDH/CS in the eye tissue of L. macrochirus was increased in September, so the ratio of anaerobic metabolism was high. The electrophoretic patterns of mitochondrial LDH were similar to those of cytosolic LDH in the eye tissues of L. macrochirus and Micropterus salmoides. LDH eye-specific $C_4$ isozyme from eye tissue was purified by preparative native-PAGE. The activities of LDH eye-specific $C_4$ isozymes in L. macrochirus and M. salmoides were reduced at concentrations greater than 0.2 mM and 0.1 mM of pyruvate, respectively. These concentrations remained at 5.2% and 15.8% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The LDH activities of eye tissues were reduced at concentrations greater than 22 mM and 24 mM of lactate, respectively, in L. macrochirus and M. salmoides. The ${K_m}^{PYR}$ of eye-specific $C_4$ was 0.088 mM in L. macrochirus and it was 0.033 mM in M. salmoides. The activities of cytosolic and mitochondrial eye-specific $C_4$ isozymes were high in ${\alpha}$-ketobutyric acid. Furthermore, the activities of eye tissue and eye-specific $C_4$ isozyme had to be measured with 0.5 mM of pyruvate and a buffer solution of pH 7.5. As a conclusion, the eye-specific $C_4$ isozyme in M. salmoides has a high affinity for pyruvate and exhibits maximum activity at a lower concentration of pyruvate and at higher concentration of lactate than that in L. macrochirus. Therefore, it seems that the energy produced by the LDH eye-specific $C_4$ isozyme in M. salmoides was used at the first stage of predatory behavior.

• Journal of Periodontal and Implant Science
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• v.29 no.3
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• pp.483-509
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• 1999

Biodegradable barrier membrane has been demonstrated to have guided bone regeneration capacity on the animal study. The purpose of this study is to evaluate the effects of cultured calvarial cell inoculated on the biodegradable barrier membrane for the regeneration of the artificial bone defect. In this experiment 35 Sprague-Dawley male rats(mean BW 150gm) were used. 30 rats were divided into 3 groups. In group I, defects were covered periosteum without membrane. In group II, defects were repaired using biodegradable barrier membrane. In group III, the defects were repaired using biodegradable barrier membrane seeded with cultured calvarial cell. Every surgical procedure were performed under the general anesthesia by using with intravenous injection of Pentobarbital sodium(30mg/Kg). After anesthesia, 5 rats were sacrificed by decapitation to obtain the calvaria for bone cell culture. Calvarial cells were cultured with Dulbecco's Modified Essential Medium contained with 10% Fetal Bovine Serum under the conventional conditions. The number of cell inoculated on the membrane were $1{\times}10^6$ Cells/ml. The membrane were inserted on the artificial bone defect after 3 days of culture. A single 3-mm diameter full-thickness artificial calvarial defect was made in each animal by using with bone trephine drill. After the every surgical intervention of animal, all of the animals were sacrificed at 1, 2, 3 weeks after surgery by using of perfusion technique. For obtaining histological section, tissues were fixed in 2.5% Glutaraldehyde (0.1M cacodylate buffer, pH 7.2) and Karnovsky's fixative solution, and decalcified with 0.1M disodium ethylene diaminetetraacetate for 3 weeks. Tissue embeding was performed in paraffin and cut parallel to the surface of calvaria. Section in 7${\mu}m$ thickness of tissue was done and stained with Hematoxylin-Eosin. All the specimens were observed under the light microscopy. The following results were obtained. 1 . During the whole period of experiment, fibrous connective tissue was revealed at 1week after surgery which meant rapid soft tissue recovery. The healing rate of defected area into new bone formation of the test group was observed more rapid tendency than other two groups. 2 . The sequence of healing rate of bone defected area was as follows ； test group, positive control, negative control group. 3 . During the experiment, an osteoclastic cell around preexisted bone was not found. New bone formation was originated from the periphery of the remaing bone wall, and gradually extended into central portion of the bone defect. 4 . The biodegradable barrier membrane was observed favorable biocompatibility during this experimental period without any other noticeable foreign body reaction. And mineralization in the newly formed osteoid tissue revealed relatively more rapid than other group since early stage of the healing process. Conclusively, the cultured bone cell inoculated onto the biodegradable barrier membrane may have an important role of regeneration of artificial bone defects of alveolar bone. This study thus demonstrates a tissue-engineering the approach to the repair of bone defects, which may have clinical applications in clinical fields of the dentistry including periodontics.

• Microbiology and Biotechnology Letters
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• v.37 no.2
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• pp.118-124
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• 2009

Esterase EM2L8 gene isolated from deep sea sediment was expressed in Escherichia coli BL21 (DE3) and the esterase activity of the cell-free extract was assayed using p-nitrophenyl butyrate-spectrophotometric method. Its optimum temperature was $40-45^{\circ}C$ and 45% activity of the maximum activity was retained at $15^{\circ}C$. The activation energy at $15-45^{\circ}C$ was calculated to be 4.9 kcal/mol showing that esterase EM2L8 was a typical cold-adapted enzyme. Enzyme activity was maintained for 6 h and 4 weeks at $30^{\circ}C$ and $4^{\circ}C$, respectively. When each ethanol, methanol, and acetone was added to the reaction mixture to 15% concentration, enzyme activity was maintained. In the case of DMSO, enzyme activity was kept up to 40% concentration. (S)-4-Chloro-3-hydroxy butyric acid is a chiral intermediate for the synthesis of Atorvastatin, a hyperlipemia drug. When esterase EM2L8 (40 U) was added to buffer solution (1.2 mL, pH 9.0) containing ethyl-(R,S)-4-chloro-3-hydroxybutyrate (38 mM), it was hydrolyzed into 4-chloro-3-hydroxy butyric acid with a rate of $6.8\;{\mu}mole/h$. The enzyme hydrolyzed (S)-substrate more rapidly than (R)-substrate. When conversion yield was 80%, e.e.s value was 40%. When DMSO was added, hydrolysis rate increased to $10.4\;{\mu}mole/h$. The plots of conversion yield vs e.e.s in the presence or absence of DMSO were almost same, implying that the reaction enantioselectivity was not changed by the addition of DMSO. Taken together, esterase EM2L8 had high activity and stability at low temperatures as well as in various organic solvents/aqueous solutions. These properties suggested that it could be used as a biocatalyst in the synthesis of useful pharmaceuticals.

• Applied Microscopy
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• v.30 no.4
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• pp.357-365
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• 2000

Paneth cells have been suggested to contribute to the elimination of excess metals into the intestinal lumen. The purpose of this study wat to investigate the changes of the zinc pools in rats subjected to functional loading with zinc salt by mean of both light and electron microscopical autometallography (AMG). Wistar rats 4 were administrated with zinc chloride (20 mg/kg body weight) intraperitoneally dissolved in 1 ml distilled water. The control group received 1 ml saline IP. After further one hour the animals were transcardially perfused with 0.4% sodium sulphide dissolved in 0.1 M PB fellowed by 3% glutaraldehyde solution for 10 minutes. Pieces of ileum were frozen with solid $CO_2$ and sectioned on a cryostat. The sections $(20{\mu}m)$ were autometallographically developed. Sections selected for EM were reembedded on top of a blank Epon block, from which ultrathin sections (100 nm) were cut. The ultrathin sections were double stained with uranyl acetate (30 min) and lead citrate (5 min), then examined under electron microscope. Studies of comparable sections from control and zinc loaded animals with the AMG selenium method gave quite different results. The control animals demonstrated a weakly positive staining in the cytoplasm of the Paneth cells. In the electron microscope the AMG silver grains were found to be located in the cytoplasm, while the electron dense secretary granules and other cell organelles were void of staining. Few AMG grains were located at the apical surface of the Paneth cells. In sections from zinc loaded rats, the AMG grains were seen in abundance in the lumen of the Lieberkuhn crypts at light microscopic levels. At EM levels the zinc revealing silver grains were located in the cytoplasm as in the controls, but much more AMG grains were shifted into the secretary granules. Furthermore, profound AMG grains were found in the lumen of the crypts and surrounding vessels. And a few grains were seen in the endothelium. The AMG technique demonstrated a pattern of AMG grains in the Paneth cells that strongly suggests a transport of zinc ions through these cells.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.315-322
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• 2006

FS11052, a novel microbial metabolite from Streptomyces spp. was identified as a small molecular substance and shown inhibition activities for the release of neurotransmitter from rat hippocampal neuron and PC12 cells. FS11052 is an inhibitor of tritiated norepinephrine ($[^3H]-NE$) release in high $K^+$ buffer solution containing ionomycin, indicating that FS11052 inhibits neurotransmitter release after the influx of $Ca^{2+}$ ions. When examined the effect of FS11052 on glucuronidase release from guinea pig neutrophils, FS11052 inhibited glucuronidase release: when treated with $5{\mu}g/ml$ of FS11052, which was not induced cellular cytotoxicity. The fact that the glucuronidase release in neutrophil and norepinephrine release in neuron was inhibited suggests the similarity in the locations and the mechanisms of FS11052 action targets. When treated with $5{\mu}g/ml$ of FS11052, $[^3H]-NE$ release and neurite extension for both rat hippocampal neurons and PC12 cells were prevented. These observations of FS11052 functioning as an inhibitor of neurotransmitter release suggest that FS11052 has an important role in synaptic transmission in neuron.

• Journal of the Korean Society of Food Science and Nutrition
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• v.16 no.1
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• pp.1-9
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• 1987

Large amount of aflatoxin $B_1(AFB_1)$ is disappeared in the presence of L-ascorbic acid(AA) in buffer solution at pH values from 1 to 7 during 5 days of Incubation at $37^{\circ}C$. $AFB_1$ was quite stable at pH's between 5 and 7 when AA was absent(control), however, $50{\sim}60%$ of APB, was degraded in its presence after 5 days. The rate of disappearance of $AFB_1$ increased with a decreasing of pH in the presence of AA, even though $AFB_1$ in the control degraded increasingly with the decrease in $pH(pH{\leq}4)$. The level of $AFB_1$, decreased as the reaction temperature increased when $AFB_1$ reacted with AA. The aflatoxin could not be detected at all after 3 days when the reaction occurred at $60^{\circ}C$, while the aflatoxin was stable at $5^{\circ}C$ thoughout the reaction period. $90{\sim}96%$ of $AFB_1$ was found to be degraded in a far when $AFB_1$ reacted with AA plus different concentrations of $CuSO_4{\cdot}5H_{2}O$, showing remarkably faster rate than the control; however, different concentrations of L-cysteine instead of $CuSO_4\;5H_{2}O$ protected the degradation of aflatoxin and no $AFB_1$ was degraded for a day and resulted in less $AFB_1$ disappeared than the control. The degradation of $AFB_1$ was dependent on AA concentration and the rate of disappearance as the concentration of AA decrease, but $AFB_1$ concentration did not influence the rate. The product formed when $AFB_1$ reacted with AA was identified to $AFB_{2a}$ by using HPLC chromatographic examinations, and by UV spectrum of $AFB_1$ reacted with AA. The disappearance of $AFB_1$ was correlated well in the appearance of $AFB_2a$. From the results, the degradation of $AFB_1$ in the presence of AA is probably due to one or more of the oxidative products of AA which was produced during the AA oxidation.

• Analytical Science and Technology
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• v.10 no.4
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• pp.282-290
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• 1997

The sorption and desorption properties of U(VI), Th(IV), Zr(IV), Cu(II), Pb(II), Ni(II), Zn(II), Cd(II) and Mn(II) ions on XAD-16-[4-(2-thiazolylazo)orcinol] (TAO) chelating resin were studied by elution method. The effect was examined with respect to overall capacity of each metal ion, separation of mixed metal ions, flow rate and concentration of buffer solution for optimum condition of sorption. The overall capacities of some metal ions on this chelating resin were 0.35nmol U(VI)/g resin, 0.49nmol Th(IV)/g resin, 0.41nmol Cu(II)/g resin, and 0.31nmol Zr(IV)/g resin, respectively. The elution order of metal ions obtained from breakthrough capacity and overall capacity at pH 5.0 was Th(IV)>Cu(II)>U(VI)>Zr(IV)>Pb(II)>Ni(II)>Zn(II)>Mn(II)>Cd(II). The group separation of mixed metal ions was possible by increasing pH in pH range 2~5 at a flow rate of 0.28mL/min. Characteristics of desorption were investigated with desorption agents such as $HNO_3$, HCl, $HClO_4$, $H_2SO_4$, and $Na_2CO_3$. It was found that 2M $HNO_3$ showed high desorption efficiency to most of metal ions except Zr(IV) ion. Also, desorption and recovery of Zr(IV) ion were successfully performed with 1M $H_2SO_4$. Recovery of trace amount of U(VI) ion from artificial sea water was over 94%. The chelating resin, XAD-16-TAO was successfully applied to group separation of rare earth metal ions from U(VI) by using 2M $HNO_3$ as an eluent.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.24-29
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• 2013

In the present study, the effects of prebiotics and prebiotics+probiotics on intestinal microflora and fermentation products were evaluated in a pig in vitro fermentation model. The substrates used in this study were iso-malto oligosaccharide (IMO), partially digested chicory-inulin (CI), raffinose (RA), and cyclodextrin (CD) as prebiotics and Lactobacillus reiteri as probiotics. For a pig in vitro fermentation, the experimental diet for growing pigs was predigested using digestive enzymes secreted by small intestine and this hydrolyzed diet was mixed with a buffer solution containing 5% fresh swine feces. The mixture was then incubated with either prebiotics or prebiotics+probiotics for 24 h. Samples were taken at 24 h, and viable counts of microflora, gas, pH, volatile organic compounds (VOCs) and short-chain fatty acid (SCFA) were analyzed. The viable count of Enterobacteriaceae was significantly decreased (p<0.001) in all treatments containing prebiotics and prebiotics+probiotics when compared to the control. However, the number of lactic acid bacteria increased in the prebiotics and prebiotics+probiotics treatment. The pH values in the fermentation fluid decreased in all treatments when compared to the control, and their effects were greater in the prebiotics+probiotics group than prebiotics group. Fermentation with prebiotics resulted in a reduction in malodorous compounds such as ammonia, hydrogen sulfide and skatole when compared to the prebiotics+probiotics group. Short-chain fatty acid production was also higher for treatment with prebiotics+probiotics than treatment with prebiotics. In conclusion, the results of this study demonstrated that fermentation with prebiotics was effective in reducing the formation of malodorous compounds and prebiotics+probiotics was effective in increasing lactic acid bacteria and SCFA and reducing the pH. Moreover, further studies will be needed to determine whether the results observed in the in vitro model would occur in pigs that ingest these prebiotics or probiotics.

• Journal of Pharmaceutical Investigation
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• v.35 no.3
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• pp.179-185
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• 2005

Acebrophylline is a compound produced by salifying ambroxol with theophylline-7 -acetic acid. After acebrophylline administration, the salt splits into these two components which feature a peculiar pharmacokinetic behavior, an adequate ambroxol and a low theophylline-7-acetic acid serum levels. The purpose of the present study was to evaluate the bioequivalence of two acebrophylline capsules, Surfolase (Hyundai Pharm. lnd. Co., Ltd.) and Burophil (Kuhnil Pharm. Co., Ltd.), according to the guidelines of the Korea Food and Drug Administration (KFDA). The release of ambroxol from the two acebrophylline formulations in vitro was tested using KP VIII Apparatus II method with various dissolution media (pH 1.2, 4.0, 6.8 buffer solution and water). Twenty eight healthy male subjects, $23.25{\pm}1.43$ years in age and $64.82{\pm}6.77$ kg in body weight, were divided into two groups and a randomized $2{\times}2$ cross-over study was employed. After two capsules containing 100 mg as acebrophylline were orally administered, blood was taken at predetermined time intervals and the concentrations of ambroxol in serum were determined using HPLC with electrochemical detector (ECD). The dissolution profiles of two formulations were similar at all dissolution media. In addition, the pharmacokinetic parameters such as $AUC_t$, $C_{max}$ and $T_{max}$ were calculated and ANOVA test was utilized for the statistical analysis of the parameters using logarithmically transformed $AUC_t$, $C_{max}$ and untransformed $T_{max}$. The results showed that the differences between two formulations based on the reference drug Surfolase, were -1.64, -3.33 and -0.92% for $AUC_t$, $C_{max}$ and $T_{max}$, respectively. There were no sequence effects between two formulations in these parameters. The 90% confidence intervals using logarithmically transformed data were within the acceptance range of log 0.8 to log 1.25 $(e.g., \;log\;0.93{\sim}log\;1.05\;and\;log\;0.88{\sim}log\;1.05$ for $AUC_t$, and $C_{max}$, respectively). Thus, the criteria of the KFDA bioequivalence guideline were satisfied, indicating Burophil capsule was bioequivalent to Surfolase capsule.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.13 no.4
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• pp.163-171
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• 1980

Recently, Bacillus has been identified as one of food poisoning bacteria especially in products of cereal foods in foreign countries. Therefore, the quantitative distribution of Bacillus cereus in market foods, its physiological characteristics, growth rate by temperature and heat resistance of its spore were examined. Thirty two samples of cooked rice, 20 samples of kimbab(cooked rice rolled with laver), 23 samples of rice cake, 13 samples of rice ana 13 samples of barley were collected from restaurents and food stores in Busan, Korea during the period from May to November in 1980. Forty samples of 101 samples submitted to the test appeared positive for Bacillus cereus showing abut $40\%$ in detection ratio. Detection ratio of Bacillus cereus was higher than $50\%$ in barley and rice, and about $30\%$ in rice products. Average Bacillus cereus content of in the samples was $2.6\times10^6/g$ in cooked rice, $2.3\times10^6/g$in kimbab, $4.9\times10^4/g$ in rice cake while that in rice and barley was about $10^3/g$. The result of biochemical tests of the bacterium was $100\%$ positive in catalase, egg yolk reaction, gelatin hydrolysis and glucose fermentation, $100\%$ negative in xylose, arabinose and mannitol oxidation, about $90\%$ positive in acetoin production, $80.0\%$ positive in nitrate reduction and citrate utilization and $55.0\%$ positive in starch hydrolysis test. Isolation ratio of Bacillus ceresus which showed haemolysis positive and starch hydrolysis negative results, was about $38\%$ in 40 strains examined. It is known that those strains has a close relation to food poisoning accident. Growth rate and generation time of Bacillus cereus isolated from the cooked rice were $0.34hr^{-1},\;2.02hr\;at\;20^{\circ}C,\;0.73hr^{-1},\;0.95hr\;at\;30^{\circ}C\;and\;0.49hr^{-1},\;1.44\;hr\;at\;40^{\circ}C$ respectively. Heat resistance value of Bacillus cereus spores suspended in phosphate buffer solution was $D_{90}=29.0min,\;D_{95}=8.7min,\;D_{98}=3.7\;min\;and\;D_{101}=2.3\;min(z=10.5)$.