• Journal of Microbiology and Biotechnology
• /
• 제14권2호
• /
• pp.231-236
• /
• 2004

The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at $26^{\circ}C$. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70％. Both 23 kDa ($\alpha$) and 62 kDa ($\beta$) subunits were recovered by using IMAC with just C-terminus tagging of the $\beta$ subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.

• 약학회지
• /
• 제28권4호
• /
• pp.207-215
• /
• 1984

Effects of Ssang Wha Tang (SWT), a blended Chinease traditional medicine, on the pharmacokinetics of sulfobromophthalein (BSP) in the rats of hepatic failure induced by carbon tetrachloride were examined. The disposition of plasma BSP in carbon tetrachloride-treated rats (Group I) and in carbon tetrachloride+SWT-treated rats (Group II) followed a three-compartment model, while those in control group followed two-compartment model. GOT, GPT level and some pharmacokinetic paramiters like plasma clearance but except distribution volume (Vdss) recovered in Group II compared to Group I. Therefore, SWT seemed to have an apparent restoring effect of hepatic function damaged by carbon tetrachloride treatment. From the fact that Vdss of BSP in Group II was considered as an one of the probable mechanisms. More intensive increase in BSP-free fraction ($f_p$) in Group II than that in Group I might also explain the increases of BSP clearance and Vdss in Group II compared to Group I. Assuming no changes in hepatic plasma flow(Q) in each group, hepatic intrinsic clearance($CL^h_{int}$) decreased in Group I did not recovered not at all in Group II. Therefore SWT seemed not to have any restoring effect of true hepaticfunction to biotransform and excrete BSP, and the apparent restoring effect of SWT might be due only to the replacement of BSP-plasma protein binding. Whether $f_p$ is actually higer in Group II than in Group I, and Q is constant in each group are being examined in our laboratory. The changes of Q, which might lead to another conculusions, also should be taken into consideration to clarify the apparent hepatorestoring effect of SWT.

• 약학회지
• /
• 제41권3호
• /
• pp.381-388
• /
• 1997

Metabolism of DWP401, recombinant juman epidermal growth factor, was examined in vivo and in vitro in rats. When $^{125}I$-labeled DWP401 was administered at a dose of 50 ${\mu}g$/kg by i.v. injection. $^{125}I$-labeled DWP401 was rapidly degraded within 30 minytes above 93%. Thin layer chromatography analysis of urine collected for 24 hr after i.v. administration of $^{125}I$-labeled DWP401 showed ohly one spot on a X-ray film which was considered as diiodo-tyrosine. This result suggests tha $^{125}I$-labeled DWP401 was completely digested into free amino acids without any specific intermediate polypeptides. About 42.1% of the administered iodine was recovered in 24 hr. For in vitro degradation study, $^{125}I$-labeled DWP401 was added to plama and tissue homogenates of rats and incubated at $37^{\circ}C$. Almost 98% of the added radioactivity recovered from the protein fraction of the liver, kidey, small intestine, stomach and spleen decreased rapidly. For examplem the recovery rates of $^{125}I$-labeled DWP401 were 58.6, 63.2, 39.9, 52.9 and 66.8% after 4hrs of incubation in respective organ homogenates.

• Journal of Photoscience
• /
• 제1권2호
• /
• pp.95-105
• /
• 1994

Experiments were carried out to investigate whether P, deficiency in detached 25 mM mannose-feeding led to a decline of the photosynthetic electron transport rates through acidification of the thylakoid lumen. With increasing mannose-feeding time, the maximal CO2 exchange rates and the maximal quantum yields of photosynthesis decreased rapidly up to 6 h by 73% then with little decrease up to 12 h. The ATP/ADP ratio declined by 54% 6 h after the treatment and then recovered to the control level at 12 h. However, the NADPH/NADP~ ratio was not significantly altered by mannose treatment. Electron transport rates of thylakoid membranes isolated from 6 h treated leaves did not change, but they decreased by 30% in 12 h treated leaves. The quenching analysis of Chl fluorescence in mannose-treated leaves revealed that both the fraction of reduced plastoquinone and the degree of acidification of thylakoid lumen remained higher than those of the control. The reduction of PSI in mannose fed leaves was inhibited due to acidification of thylakoid lumen (high qE). The reduction of primary quinone acceptor of PSII was inhibited by mannose feeding. Mannose treatment decreased the efficiency of excitation energy capture by PSII. Fo quenching was induced when treated with mannose more than 6 h, and had a reverse linear correlation with (Fv)m/Fm ratio. These results suggest that Pi deficiency in Chinese cabbage leaves reduce photosynthetic electron transport rates by diminishing both PSII function and electron transfer from PSII to PSI through acidification ofthylakoid lumen, which in turn induce the modification of photosynthetic apparatus probably through protein (de)phosphorylation.

• 식물조직배양학회지
• /
• 제24권4호
• /
• pp.197-201
• /
• 1997

Plant seed oil-bodies or oleosomes ate the repository of the neutral lipid stored in seeds. These organelles in many oilseeds may comprise half of the total cellular volume. Oleosomes are surrounded by a half-unit membrane of phospholipid into which are embedded proteins called oleosins. Oleosins are present at high density on the oil-body surface and after storage proteins comprise the most abundant proteins in oilseeds. Oleosins are specifically targeted and anchored to oil-bodies after co-translation on the ER. It has been shown that the amino-acid sequences responsible for this unique targeting reside primarily in the central hydrophobic tore of the oleosin polypeptide. In addition, a signal-like sequence is found near the junction of the hydrophobic domain and ann N-terminal hydrophilic / amphipathic domain. This "signal" which is uncleaved is also essential for correct targeting. Oil-bodies and their associated oleosins may be recovered by floatation centrifugation of aqueous seed extracts. This simple partitioning step results in a dramatic enrichment for oleosins in the oil-body fraction. In the light of these properties, we reasoned that it would be feasible to create fusion proteins on oil-bodies comprising oleosins and an additional valuable protein of pharmaceutical or industrial interest. It was further postulated that if these proteins were displayed on the outer surface of oil-bodies, it would be possible to release them from the purified oil-bodies using chemical or proteolytic cleavage. This could result in a simple means of recovering high-value protein from seeds at a significant (i.e. commercial) scale. This procedure has been successfully reduced to practice for a wide variety of proteins of therapeutic, industrial and food no. The utillity of the method will be discussed using a blood anticoagulant, hirudin, and industrial enzymes as key examples.

• 고려인삼학회:학술대회논문집
• /
• 고려인삼학회 1987년도 Proceedings of Korea-Japan Panax Ginseng Symposium 1987 Seoul Korea
• /
• pp.29-37
• /
• 1987

This study was devised to observe the cytotoxlc activities of petroleum-ether extract of Panax ginseng root(crude Gx) and its partially purified fraction from silicon acid column chromatography(7:3 CX) against sarcoma-180(5-180) and Walker carcinosarcoma 256(Walker 256) in vivo, and murine leukemic lymphocytes(L1210) and human rectal cancer cell(HRT-18) and human colon cancer cells(HT-29 and HCT-48) in vitro . Each cell-line was cultured in medium containing serial concentrations of the crude Gx or 7:3 Gx in vitro. A highly lipid soluble compound in the extract of Panax ginseng root was cytocidal to murine leukemic cells and human colon and rectal cancer cells in vitro In the meantime, ginseng saponin derivatives did not cytotoxic effects at its corresponding concentration. The growth rates of the cancer cells in medium containing ginseng extracts were inhibited gradually to a significant degree roughly in proportion to the increase of the extract concentration. The cytotoxic activity of 7:3 Gx was about 3 times more potent than that of crude Gx, one unit of cytotoxic activity against L121f cells being equivalent to 2.54$\mu\textrm{g}$ and 0.88 $\mu\textrm{g}$ for the crude Gx and 7:3 Gx, respectively. The Rf value of the active compound on silica -gel thin layer chromatography with petroleum-ether/ethyl ether/acetic acid mixture (90:10:1, v/v/v) as a developing solvent was 0.23. The survival times of mice inoculated with S-180 cells were extended about 1.5 to 2 times by the 7:3 Gx treatment compared with their control group. The significantly decreased hemoglobin values of rats after inoculation with Walker 256 were recovered to normal range by oral administration of the crude Gx. The synthetic levels of protein, DNA and RNA in human colon and rectal cancer cells were significantly diminished by treatment with the crude Gx, which can explain a part of the origin of its anticancer activity.

• 한국축산식품학회지
• /
• 제20권2호
• /
• pp.159-165
• /
• 2000

Glycomacropeptide(GMP) was purified from cheese whey which is obtaining as a byproduct in cheese producing. Cheese whey was first concentrated 10 times with a ultrafiltration aparratus, and then heated at 95℃ for 5 min. The concentrated fraction was centrifuged at 20,000×g for 30 min to remove fat layer. The supernatant layer enriched GMP protein was fractionated by ion exchange chromatography on DEAE-Sepharose Fast Flow column. GMP was bound to DEAE resin and eluted with 0.1∼0.25 M NaCl when using a linear NaCl gradient from 0 M to 0.5 M. The purified GMP gave a single band of 24 kDa which seems to be trimer molecular weight in SDS-PAGE, and migrated to the same molecular weight with control GMP obtained commercially. Its amino acid composition were consistent with that of standard GMP. About 0.71 g of GMP was recovered from 1 L of cheese whey. These results indicate that glycomacropeptide could be simply purified from cheese whey by using ultrafiltration and DEAE column chromatography. 본 연구에서는 치즈 제조시의 부산물인 치즈 유청으로부터 glycomacropeptide(GMP)를 정제하려고 하였다. 치즈 유청을 한외여과 장치를 이용하여 10배로 농축하여 공시시료로 하였고, 95℃에서 5분간 가열한 후 20,000rpm에서 30분간 원심분리하였다. 원심분리 후 혼입된 지방층을 제거하였고, GMP를 포함하고 있는 상청액은 DEAE-Sepharose Fast Flow 크로마토그래피 칼럼에 주입하였다. GMP는 DEAE 수지의 관응기에 결합하였으며, 0에서 0.5M의 NaCl 직선농도 구배를 실시하였을 때 약 0.1∼0.25M에서 용출되었다. 정제한 GMP는 SDS-PAGE에서 단일한 band를 나타냈으며, 분자량은 24kDa으로 trimer 형태로 상업적으로 제공받은 대조 GMP와 같은 분자량 위치로 영동하였다. 정제한 GMP의 아미노산 조성은 대조GMP의 결과와 비교하였을 때 Ser, Val이 약간 적었으며 Gly은 반대로 2배의 함량을 나타냈으나 전체적인 조성비에서는 거의 일치하였다. 단백질의 회수율은 유청 1L에서 약 0.71g의 GMP를 효과적으로 분리한 것으로 나타났다. 이 결과로부터 한외여과와 DEAE-Sepharose Fast Flow chromatography를 이용하여 GMP를 산업적으로 대량 정제할 수 있는 것이 확인되었다.

• 대한수의학회지
• /
• 제15권2호
• /
• pp.187-198
• /
• 1975

Although considerable research has been done on the blood chemistry of domestic animals, little work has been made of the changes associated with age. Moreover, the records about physiology of the goat were not much available in Korea, and a comprehensive survey of the blood values of the Korean native goat has not been made. The object of the present investigation was to make good this deficiency and to suggest standards for the blood chemical values of Korean native goats from birth to maturity. The goats were kept under average farming conditions in Korea. Blood samples were collected from the jugular vein at birth, at one and four days, at one, two, three and four weeks, and at two, three, six, nine and twelve months of age. The results obtained in this work were summarized as follows: 1. The highest concentration of serum glucose was observed at birth and it decreased gradually until three months of age, showing a steady state thereafter. 2. The concentration of total serum protein was a little higher at one day of age than at birth and showed a steady state until seven days of age; then it decreased slightly at two weeks of age and recovered at three weeks of age and showed a steady state until twelve months of age. The concentration of serum albumin was increased a little at one day of age and revealed a steady state thereafter. The concentration of serum globulin showed three phases - a fall during the first two weeks to reach minimum, a rise to the fourth week, and a fall at two months of age, showing a steady state thereafter. The lowest value of albumin fraction was observed at birth, then the value increased gradually until three months of age and revealed a stealer state thereafter. The highest value of globulin fraction was observed at birth, then the gradual decrease of the value was seen until three months of age; thereafter the value showed a steady state. 3. Albumin/globulin ratio was the minimum at birth, then it increased to reach a maximum at two weeks of age and decreased a little thereafter. 4. The concentration of total serum cholesterol showed a gradual increase during the first three months and fell to reach adult revel at six months of age. 5. Urea nitrogen in serum decreased during the first week to reach a minimum, then it increased at three months of age, and showed a fell to mature level at six mouths of age. 6. The concentration of serum creatinine was not affected by age. 7. The concentration of total serum calcium was a little higher during the first two weeks than the other period during the first year of life. 8. The concentration of serum inorganic phosphorus showed a gradual rise to reach a maximum and a gradual fall to reach adult level at nine months of age. 9. No significant sex differences of serum chemical values were recognized.

• 동의생리병리학회지
• /
• 제18권6호
• /
• pp.1652-1660
• /
• 2004

Apoptosis of vascular smooth muscle cells(VSMCs) is essential in atherogenesis, being a factor that modulates its early progression rather than a terminal event in the course of the disease. Various stimuli, including oxide lipoproteins, altered hemodynamic stress and free radical, can induced VSMCs apoptosis in vitro. The protective effects of Sophorae Radix (SR) on apoptotic cell death induced by H₂O₂ were investigated in VSMCs. The viability of VSMCs was markedly decreased by H₂O₂. Sophorae Radix protected the H202-induced apoptotic death of VSMCs, which was characterized as nuclear fragmentation and increase of sub-G0/G1 fraction .. Sophorae Radix decreased the activation of caspase-3 like protease induced by H₂O₂ and recovered control level from H202-induced PARP, Bak, Bcl-XL and mitochondrial membrane potential. These results suggest that Sophorae Radix protected VSMCs apoptotic death induced by H₂O₂ via inactivation of caspase-3 and modulation of mitochondrial function. Also, the expression profile of proteins by using two-dimensional (2-D) gel electrophoresis was screened. Future investigations will need to explore the use of an anti atherosclerotic therapy of Sophorae Radix, which relies on inhibition of the proapoptotic activation of the vascular smooth muscle cells.

• 한국임상약학회지
• /
• 제21권3호
• /
• pp.256-269
• /
• 2011

Omega-3 fatty acids are a specific type of unsaturated fat that the body cannot manufacture on its own, so they must be obtained from food which is essential fatty acids (EFAs). Omega-3 fatty acids consist of three types which are a-Linolenic Acid (ALA), Eicosapentaenoic (ELA), and Docosahexaenoic Acid (DHA). Especially, EFAs help to prevent skin and hair drying, acne, eczema, prevention from allergies, brittle nails, rashes, and tiny lumps. The aim of this study is to investigate improvement and protection for hair damaged by chemical treatment with omega-3 formulated shampoo. We selected virgin hair sample and divided into two groups for bleaching once and three times and then damaged hair by changing the number of hair bleaching (twice with interval of 15 minutes). Each bleached hair was treated by five different kinds of shampoo (Control, Horse shampoo, DHA shampoo, EPA shampoo, Omega-3 shampoo mixture). Apart from this, EPA/DHA 2, 5, 8, 10 and 12% shampoo were prepared and treated to hair for comparing rate of progress in damaged hair. To quantify improved condition of damaged hair, we performed Scanning Electron Microscope (SEM) for ultrastructure of damaged hair fraction, measurement of thickness change and BCA Protein Assay for recovery rate of damaged hair. The moisture in hair was measured by Thermal analysis machine. In results, we observed the particle of hair surface damaged by bleaching treatment were well improved with treatment with EPA and DHA shampoo. Also, quantity of protein was lowered with higher concentration of EPA & DHA i.e., 8 and 12 % then compared with horse oil shampoo in three times treatment group. It shows that bleached hair have been recovered by treating rapidly and get protective coat. In conclusion, EPA and DHA shampoo improved damaged hair, especially with EPA / DHA 12% shampoo. Also, EPA shampoo could protect the damaged hair depending on increasing concentration of EPA. Therefore, we conclude omega-3 shampoo could make damaged hair protect and get healthy hair environment.