• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.196-202
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• 2006

Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However the most troublesome problem of lamivudine treatment is the emergence of lamivudine-resistant strains with amino acid substitution in the YMDD motif of DNA polymerase gene during the treatment. The aim of this study was to determine the mutation of YMDD motif (codon 552) and codon 528 in chronic HBV patients with lamivudine therapy using PCR-direct sequencing and to investigate the relationship between lamivudine mediated HBV mutation and HBeAg. HBV DNA was extracted from serum samples of HBV patients and amplified by nested PCR with two sets of primer pairs selected in HBV DNA polymerase gene. Amplified PCR product was analyzed by 2% agarose gel electrophoresis and direct sequencing. HBV mutation was detected in 124 out of 207 samples (60%). Single mutation was 50.8% for M552I, 43.5% for M552V, 5.7% for M552I/V and the L528M mutation was 67.0%. Double mutation was 43.6% for M552V/L528M, 33.1% for M552I/L528(wild type), 17.7% for M552I/L528M and 5.6% for M552I/V/L528M. Serine mutation at YMDD motif (M552S) was not found and the L528M mutation frequently accompanied M552V type. In this study, the typical difference of frequencies for HBV mutation depending on HBeAg was not found. Moreover, the PCR-direct sequencing method used in this study might be a powerful tool for the mutation study in clinical reference laboratories with high volume.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.22 no.1
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• pp.135-166
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• 1977

Some biochemical features of varietal variation in seed protein and their implications for soybean breeding for high protein were pursued employing 86 soybean varieties of Korea, Japan, and the U.S.A. origins. Also, studied comparatively was the temporal pattern of protein components accumulation during seed development characteristic to the high protein variety. Seed protein content of the 86 soybean varieties varied 34.4 to 50.6%. Non-existence of variety having high content of both protein and oil, or high protein content with average oil content as well as high negative correlation between the content of protein and oil (r=-0.73$^{**}$) indicate strongly a great difficulty to breed high protein variety while conserving oil content. The total content of essential amino acids varied 32.82 to 36.63% and the total content of sulfur-containing amino acids varied 2.09 to 2.73% as tested for 12 varieties differing protein content from 40.0 to 50.6%. The content of methionine was positively correlated with the content of glutamic acid, which was the major amino acid (18.5%) in seed protein of soybean. In particular, the varieties Bongeui and Saikai ＃20 had high protein content as well as high content of sulfur-containing amino acids. The content of lysine was negatively correlated with that of isoleucine, but positively correlated with protein content. The content of alanine, valine or leucine was correlated positively with oil content. The seed protein of soybean was built with 12 to 16 components depending on variety as revealed on disc acrylamide gel electrophoresis. The 86 varieties were classified into 11 groups of characteristic electrophoretic pattern. The protein component of Rm=0.14(b) showed the greatest varietal variation among the components in their relative contents, and negative correlation with the content of the other components, while the protein component of Rm=0.06(a) had a significant, positive correlation with protein content. There was sequential phases of rapid decrease, slow increase and stay in the protein content during seed development. Shorter period and lower rate of decrease followed by longer period and higher rate of increase in protein content during seed development was of characteristic to high protein variety together with earlier and continuous development at higher rate of the protein component a. Considering the extremely low methionine content of the protein component a, breeding for high protein content may result in lower quality of soybean protein.n.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.177-186
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• 1986

This study was designed to evaluate the partially purified antigens which were fractionated from crude extract of Paragonimus westermani and to monitor the enzyme-linked immunosorbent assay (ELISA) in experimental cat paragonimiasis during the course of infection as well as before and after chemotherapy. Crude extract of 6-month-old adult P. westermani was fractionated to 5 antigens by successive applications of ammonium sulfate precipitation, ion exchange chromatography and gel filtration. And the cats, 10 in each group, were infected with 60, 30, 15, and 5 metacercariae, then the half of each group was treated with praziquantel 2 times in one day of 100mg per kilogram of weight on 150 days after the infection. Sera were collected every 10 days. ELISA was performed with the concentration of $2{\mu}g/ml$ antigen, 100 times diluted sera and 1,000 times diluted alkaline phosphatase conjugated anti-cat IgG. The results were as follows: 1. Absorbance by ELISA with proteins precipitated by differential concentration of ammonium sulfate was the highest at $51{\sim}65%$ precipitate (PA2), followed by $0{\sim}50%$ precipitate (PAl), $66{\sim}80%$ precipitate (PA3), and $81{\sim}90%$ precipitate (PA4). Unprecipitated protein over 90% ammonium sulfate (PA5) showed the lowest antigenicity. 2. Fractionation of PA1, PA2, and PA3 through the DEAE-cellulose column did not differentiate the antigenic proteins. 3. By passing through the Sephadex G-200 column, PA1 and PA2 were fractionated to high molecular weight proteins and those of low molecular weight which showed high absorbance by ELISA (PA1-I, II and PA2-I, II). But PA3 was shown to have a fraction of high molecular weight proteins (PA3-I) which showed high antigenicity. 4. SDS-polyacrylamide gel electrophoresis of PA1-I, P A1-II, PA2-I, PA2-II, PA3-I, and crude extract was performed. Fraction PA1-I was composed of proteins which had the molecular weight of 270 kilodaltons(KD) to 196 KD; of them 220KD protein was major band. Fraction PA2-I was composed of $255{\sim}225\;KD$, and PA3-I, $255{\sim}240\;KD$, respectively. Fraction PA1-II and fraction PA2-II consisted of 30 KD proteins. 5. Absorbance by ELISA began to increase within $10{\sim}20$ days after the infection and reached the highest on $140{\sim}180$ days, then made plateau thereafter. 6. Absorbance by ELISA decreased after praziquantel treatment. In 60 metacercariae infection group, the absorbance had been decreasing, but remained within the positive range during observation period, while those of 30, 15, and 5 metacercariae infection groups turned to negative range. 7. Fraction PA1-II showed the highest antigenicity in ELISA, then fraction PA2-I, fraction PA1-I, fraction PA2-II, fraction PA3-I and crude extract followed. In early phase of infection, the absorbance of fraction PA1-II showed more rapid increase than those of the other fractions and it came to positive range at $20{\sim}30$ days after infection.

• Tuberculosis and Respiratory Diseases
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• v.46 no.2
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• pp.241-250
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• 1999

Background: Persistent nonproductive cough is a major adverse effect encountered with ACE inhibitor treatment and the most frequent reason for withdrawal of the drug. The mechanism of cough was postulated to be associated with accumulation of bronchial irritants which are substrates of ACE. It has been speculated that occurrence of this adverse effect is genetically predetermined ; in particular, variants of the genes encoding ACE. To investigate this relationship, we determined ACE gene Insertion/Deletion polymorphism in subjects with and without a history of ACE inhibitor-induced cough. Methods: Among the 339 patients with ACE inhibitor treatment, subjects who developed cough that resolved when not taking medication were designated to cough group and other subjects who did not complain cough were designated to non-cough group. Clinical characteristics of the patients were collected by review of medical records. ACE genotypes were determined by PCR amplification of DNA from peripheral blood and agarose gel electrophoresis. Results: 37 patients complained of dry cough(cough group) and 302 patients did not complained of cough(non-cough group). The incidence of ACE inhibitor induced dry cough was 10.9%. There was a preponderance of females in the cough group (M : F=24.3% : 75.7%) compared to the non-cough group (M : F=49.7% : 50.3%, p=0.004). There was no significant difference in mean age, underlying diseases, and kinds and frequencies of ACE inhibitors and their mean dosage between the both groups. ACE genotypic frequencies were I/I : I/D : D/D=16.2% : 18.9% : 64.9% in the cough group and 18.9% : 18.2% : 62.9% in the non-cough group which showed no significant difference between the both groups(p=0.926). Allelic frequencies were I : D = 25.7% : 74.3% and 28.0% : 72.0% in the cough and non-cough group respectively and the difference was not significant(p = 0.676). Conclusion: The incidence of ACE inhibitor-induced cough are 10.9%, and women are more susceptible to ACE inhibitor-induced cough. ACE inhibitor-induced dry cough is not associated with ACE gene Insertion/Deletion polymorphism.

• Journal of Life Science
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• v.20 no.2
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• pp.260-268
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• 2010

The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Channa argus were purified and characterized by biochemical, immunochemical and kinetic methods. The activity of LDH in skeletal muscle was the highest at 380.4 units and those in heart, eye and brain tissues were 13.4, 3,5 and 5.4 units, respectively. Citrate synthase (EC 4.1.3.7, CS) activity in heart tissue was the highest at 20.7 units. LDH/CS in skeletal muscle, heart, eye and brain tissues were 172.9, 0.6, 0.32 and 0.47. Protein concentration in skeletal muscle tissue was 14.7 mg/g and specific activities of LDH in skeletal muscle, heart, eye and brain tissues were 25.88, 0.79, 0.31 and 1.38 units/mg, respectively. Therefore, skeletal muscle tissue was anaerobic and heart tissue was aerobic. The LDH isozymes in tissues were identified by polyacrylamide gel electrophoresis, immunoprecipitation and Western blot with antiserum against $A_4$, $B_4$, and eye-specific $C_4$. LDH $A_4$, $A_3B$, $A_2B_2$. $AB_3$ and $B_4$ isozymes were detected in every tissue, $C_4$, $AC_3$, $A_2C_2$ and $A_3C$ were detected in eye tissue, and $A_3C$ was found in brain tissue. LDH $A_4$, $A_3B$, $A_2B_2$, $AB_3$, $B_4$, eye-specific $C_4$ isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH $A_4$ isozyme was purified in the fraction from elution with $NAD^+$ containing buffer of affinity chromatography. Eye-specific $C_4$ isozyme was eluted right after $A_4$, after which $B_4$ isozyme was eluted with plain buffer. As a result, one part of molecular structures in $A_4$, $B_4$ and eye-specific $C_4$ were similar, but were different from each other in $B_4$ and $C_4$. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The activity of LDH $A_4$, $A_2B_2$, $B_4$, and eye-specific $C_4$ isozymes remained at 39.98, 21.28, 19.67 and 16.87% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The $Km^{PYR}$ values were 0.17, 0.27 and 0.133 mM in $A_4$, $B_4$ and eye-specific $C_4$ isozymes, respectively. The optimum pH of LDH $A_4$, $B_4$, eye-specific $C_4$, $A_2B_2$, $A_3B$, and $AB_3$ were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The $A_4$ and heterotetramer isozymes stabilized a broad range of pH. Especially, LDH activities in skeletal muscle tissue were high, resulting in a high degree of muscle activity.LDH metabolism in eye tissue seems to be converted faster from pyruvate to lactate by eye-specific $C_4$ isozyme as eye-specific $C_4$ have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by $A_4$ isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of $C_4$ isozyme were different according to the ecological environmental and feeding capturing patterns.

• Korean Journal of Agricultural Science
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• v.20 no.1
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• pp.56-67
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• 1993

To applying of genetic markers of milk proteins as dairy cow registration and selection aids for genetic improvement, genopypes controlling the 4 milk protein loci, ${\alpha}S1$-casein (${\alpha}S1$-CN), ${\beta}$-casein(${\beta}$-CN), ${\kappa}$-casein(${\kappa}$-CN), and ${\beta}$-lactoglobulin(${\beta}$-LG), from a total of 159 Holstein lactating cows reared at National Animal Breeding Station in 1992 were detected by polyacrylamide gel(PAGE) electrophoresis, and associations between genetic polymorphisms of milk proteins and milk compositions were analyzed. The observed distribution of phenotypes for ${\alpha}S1$-CN, ${\beta}$-CN, ${\kappa}$-CN and ${\beta}$-LG were agreement with those expected under the assumption of genetic equilibrium. The observed genotypic frequencies of the ${\alpha}S1$-CN BB, ${\beta}$-CN AA, ${\kappa}$-CN AA and ${\beta}$-LG AB genotypes were founded to be very high as 79.87%, 84.28%, 71.70% and 49.10%, respectively. Gene frequencies were 0.899 and 0.101 for ${\alpha}S1-CN^B$ and ${\alpha}S1-CN^C$, 0.921 and 0.079 for ${\beta}-CN^A$ and ${\beta}-CN^B$, 0.837 and 0.163 for ${\kappa}-CN^A$ and ${\kappa}-CN^B$, 0.378 and 0.622 for ${\beta}-LG^A$ and ${\beta}-CN^B$. According to the results of analysis of variance, the genotypes of the ${\alpha}S1-CN$, ${\beta}-CN$, ${\kappa}-CN$ and ${\beta}-LG$ were significantly difference for fat, protein and total solid percentage in milk compositions. On milk compositions, the ${\kappa}$-CN BB genotype was very high fat and protein percentage more than ${\kappa}$-CN AA and AB genotypes, and ${\beta}$-LG AA genotype was very high fat percentage more than ${\beta}$-LG AB and BB genotype at 5% level of significant difference, respectively. As a consequence, the fat and protein percentage may be improved to select to ${\kappa}$-CN BB and ${\beta}$-LG AA genotypes.

• Korean Journal of Agricultural Science
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• v.12 no.1
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• pp.1-16
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• 1985

This experiment was conducted to study sex expression and subsequent changes in protein and peroxidase after three cultivars of cucumber plants were treated with ethephon and gibberellin. The three cultivars of cucumber used in this study included 'Sayeup' (monoecious type), 'Sinrokdadaki' (gynoecious type), and 'Seonghowon' (intermediate type). The ethephon at 250 ppm and gibberellin at 100 ppm were treated at the 2-leaf and 4-leaf stages, and subsequent sex expression and changes in protein contents, peroxidase activities and isoperoxidase banding patterns by disc polyacrylamide gel electrophoresis were studied. The results can be summarized as follows: 1. Ethephon treatment slightly increased number of pistillate flowers and significantly decreased number of staminate flowers in the three cultivars, while gibberellin treatment significantly increased number of staminate flowers in both gynoecious 'Sinrokdadaki' and intermediate 'Seonghowon' and did not increase number of staminate flowers in monecious 'Sayeup' 2. There were some differences among three cultivars in protein contents, protein banding and isoperoxidase banding patterns of seeds and germinating seeds. However, it was not obvious to differentiate monoecious from gynoecious cultlvars by these characters. 3. Protein contents in the leaves. and stem apex after ethephon and gibberellin treatment increased gradually at the 2-leaf stage, but decreased at the 4-leaf stage. Protein contents in stem apex at the 4-leaf stage without treatment were much higher in 'Sinrokdadaki' and 'Seonghowon' than in monoecious 'Sayeup'. Protein contents in the stern apex at the 4-leaf stage were increased in the ethephon-treated monoecious 'Sayeup' and decreased in 'Sinrokdadaki' and 'Seonghowon' compared with untreated plants. 4. Peroxidase activities in the leaves and stem apex gradually decreased at the 2- leaf stage, but increased at the 4-leaf stage. Peroxidase activities in stern apex at the 4-leaf stage were significantly increased by ethephon treatment. 5. The number of protein bands in the three cuitivars after treatment gradually decreased in leaves and stem apex at the 2-leaf stage, but increased in the 4-leaf stage. The protein banding pat terns of stern apex of the ethephon-treated monoecious 'Sayeup' at the 4-leaf stage were gradually shifted to the banding.

• The Journal of Natural Sciences
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• v.4
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.

• 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.

• Radiation Oncology Journal
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• v.19 no.2
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• pp.153-162
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• 2001

Purpose : The mechanical insights of death of cancer cells by ionizing radiation are not of yet clearly defined. Recent evidences have demonstrated that radiation therapy may induce cell death via activation of signaling pathway for apoptosis in target cells. This study is designed whether ionizing radiation may activate the signaling cascades of apoptosis including caspase family cysteine pretenses, $Bcl_2/Bax$, cytochrome c and Fas/Fas-L in target cells. Materials and Methods : HL-60 cells were irradiated in vitro with 6 MV X-ray at dose ranges from 2 Gy to 32 Gy. The cell viability was tested by M assay and the extent of apoptosis was determined using agarose gel electrophoresis. The activities of caspase proteases were measured by proteolytic cleavages of substrates. Western blot analysis was used to monitor PARP, Caspase-3, Cytochrome-c, Bcl-2, Bax, Fas and Fas-L. Results : Ionizing radiation decreases the viability of HL-60 cells in a time and dose dependent manner. Ionizing radiation-induced death in HL-60 cells is an apoptotic death which is revealed as characteristic ladder-pattern fragmentation of genomic DNA over 16 Gy at 4 hours. ionizing radiation induces the activation of caspase-2, 3, 6, 8 and 9 of HL-60 cells in a time-dependent manner. The activation of caspase-3 pretense is also evidenced by the digestion of poly (ADP-ribose) polymerase and procaspase 3 with 16Gy ionizing irradiation. Anti-apoptotic Bcl2 expression is decreased but apoptotic Bax expression is increased with mitochondrial cytochrome c release in a time- dependent manner. In addiiton, expression of Fas and Fas-L is also increased in a time dependent manner. Conclusion : These data suggest that ionizing radiation-induced apoptosis is mediated by the activation of various signaling pathways including caspase family cysteine proteases, $Bcl_2/Bax$, Fas and Fas-L in a time and dose dependent manner.