The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.
A quantitative analytical method has been established for the measurement of inosine 5'-monophosphate dehydrogenase (IMPDH) activity in human peripheral blood mononuclear cells (PBMCs) by ion-pair reversed-phase high performance liquid chromatography equipped with ultraviolet detection (HPLC/UV). IMPDH is a ${\beta}$-nicotinamide adenine dinucleotide hydrate (NAD+)-dependent dehydrogenase in which the enzyme converts inosine 5'-monophosphate (IMP) into xanthosine 5'-monophosphate (XMP). Its activity was measured by quantifying a HPLC chromatogram corresponding to XMP produced during the incubation of lysed PBMCs with IMP as a substrate and $NAD^+$ as a coenzyme. XMP produced was detected at a wavelength of 260 nm. The mobile phase was composed of a mixture of 37 mM potassium dihydrogen phosphate containing 7 mM tetra-n-butylammonium hydrogen sulfate adjusted to pH 5.5 and methanol (85:15, v/v) with a flow rate of 1 mL/min. The calibration curve was linear ($r^2$=0.999999) in the range of $0.2-50.0\;{\mu}M$ and the limit of quantification (LOQ) was $0.2\;{\mu}M$. The intra- and inter-day precisions were between 0.88-1.47% and 0.85-5.24%, respectively. The intra- and inter-day accuracies were between 98.74-99.99% and 99.95-101.65%, respectively. IMPDH activity in 11 Korean healthy volunteers ranged from 18.29 to 36.60 nmol/h/mg protein (mean = $27.70{\pm}6.28\;nmol/h/mg$ protein).
Jurassic granite from Geochang was analysed with respect to the characteristics of the rock cleavage. The phases of distribution of microcracks were well evidenced from the enlarged photomicrographs(${\times}6.7$) of the thin section. In this study, the length - cumulative frequency diagrams were used for expressing the distribution characteristics of microcrack. The diagrams for the six directions were arranged in the magnitude of density(${\rho}$). These diagrams show an order of H2 < H1 < G2 < G1 < R2 < R1 from the related chart. Among six diagrams, the diagram for hardway 2(H2) occupies the lowermost region on the left. On the contrary, the diagram for rift 1(R1) occupies the uppermost region on the right. Curve patterns of the two diagrams change from uniform to exponential distribution type in accordance with the increased density. The overall distribution characteristics of the diagrams were well evidenced from the magnitude of the exponent(${\lambda}$) and length of line oa related to the exponential straight line. The magnitude of exponent governing the values of slope(${\theta}$) is inversely proportional to the values of microcrack parameters such as number(N), length(L) and density. On the contrary, length of line oa is directly proportional to the values of the above three parameters. Above microcrack parameters related to the order of arrangement of diagrams show an order of hardway(H1 + H2) < grain(G1 + G2) < rift(R1 + R2). The distribution characteristics of progressive variation are found among the six diagrams. The order of arrangement of the diagrams indicates a relative magnitude of the rock cleavage. Meanwhile, the parameters such as slope, exponent, density and length of line oa were arranged in an order of H2 < H1 < G2 < G1 < R2 < R1. The variation curves of a smooth quadratic function are shown from the related chart. From the correlation chart between density and the above parameters, a common regularity following power-law correlation function was derived. Finally, the analysis for the rock cleavage was conducted through the combination between the diagram and microcrack parameter. This type of combination contribute to the progressivity in evaluation for the rock cleavage.
Seo, Geun-Young;Park, Hyo-Jin;Jang, Sung-Geun;Park, Young-Hyun
Journal of the Korean Society of Food Science and Nutrition
/
v.35
no.8
/
pp.979-984
/
2006
Although iron is essential for many physiological processes, excess iron can lead to tissue damage by promoting the generation of reactive oxygen species (ROS). There is increasing evidence that ROS might play an important role in the pathogenesis of cardiovascular disease. However, the effects of iron excess on platelet function and the thrombotic response to vascular injury are not well understood. We examined the effects of iron excess-induced oxidative stress and the antioxidants on platelet aggregation. Oxidative stress was accessed by either free iron $(Fe^{+2})$ or hydrogen peroxide $(H_2O_2)$, as well as their combination on washed rabbit platelets (WPs) in vitro. When WPs were stimulated with either $Fe^{+2}$ alone or a subthreshold concentration of collagen, which gave an aggregatory curve with a little effect, and a dose dependent increase in platelet aggregation was observed by increasing concentrations of $Fe^{+2}$ with $H_2O_2$. This aggregation was associated with the iron-catalyzed formation of hydroxyl radicals from $H_2O_2$, and were inhibited by NAD/NADP (proton acceptor), catalase $(H_2O_2\;scavenger)$, tiron (iron chelator), mannitol (hydroxyl radical scavenger), and indomethacin (cyclooxygenase inhibitor), but not by NADH/NADPH (proton donor), superoxide mutase, and aspirin. However, NADH/NADPH, an essential cofactor for the antioxidant capacity by the supply of reducing potentials, showed the effect of an enhanced radical formation, suggesting a role for NADH/NADPH-dependent oxidase. These results suggest that iron $(Fe^{+2})$ can directly interact with washed rabbit platelets and this aggregation be mediated by OH formation as in the Fenton reaction, inhibited by radical scavengers.
Choi, Eun Jung;Kang, Sung Tae;Jung, So Young;Shin, Jae Min;Jang, Min Su;Lee, Sang Me;Kim, Jung Hun;Chae, Young Zoo
Korean Journal of Food Science and Technology
/
v.44
no.6
/
pp.658-665
/
2012
A survey of zearalenone contamination was conducted on cereal-based products by using an immunoaffinity column with LC-MS/MS. The calibration curve showed good lineality, with correlation coefficients ($R^2$) of 0.999 in the concentration range from 1 to 250 ng/mL. The limits of detection and quantification were approximately $0.3{\mu}g/kg$ and $1.0{\mu}g/kg$, respectively. The recoveries in the barley tea, Misutgaru and snack ranged from 73.6-107.8%. Zearalenone was detected in 10 samples (11.2% incidence). The highest zearalenone contamination level was $29.7{\mu}g/kg$ in the Misutgaru. This survey was conducted with uncertainty of measurement. The expanded uncertainty for zearalenone was estimated to be $44.9{\pm}5.0{\mu}g/kg$ (k=2, 95% confidence level) and $128.7{\pm}7.9{\mu}g/kg$ (k=2, 95% confidence level) for barley tea, $30.7{\pm}5.8{\mu}g/kg$ (k=2, 95% confidence level) and $173.7{\pm}14.9{\mu}g/kg$ (k=2.26, 95% confidence level) for Misutgaru, and $37.2{\pm}7.4{\mu}g/kg$ (k=2.31, 95% confidence level) and $151.0{\pm}10.4{\mu}g/kg$ (k=2, 95% confidence level) snack at the level of $41.7{\mu}g/kg$ and $166.7{\mu}g/kg$, respectively.
Park, Ju-Hun;Sung, Ki-Seung;Kim, Sung-Soo;Shim, Gun-Sub;Han, Chan-Kyu
Journal of the Korean Society of Food Science and Nutrition
/
v.41
no.5
/
pp.630-637
/
2012
This study was performed to investigate the effects of puffed and fermented red ginseng on blood glucose-related biomarkers in streptozotocin-induced diabetic rats. Male Sprague-Dawley diabetic rats were orally injected with 0.85% NaCL as a diabetic control (DC), 300 mg/kg general red ginseng (RG), 300 mg/kg puffing red ginseng fermented by mixed strain culture of $Bifidobacterium$$breve$ and $Lactobacillus$$delbrueckii$ (BL), and 300 mg/kg puffing red ginseng fermented by $Enterococcus$$faecalis$ (EF) for 5 weeks. The blood glucose level of group BL was significantly lower maintained than in groups DC and RG for the experimental period (p<0.05). It was also significantly lower than in groups DC, RG, and EF at the 5th week (p<0.05). In the oral glucose tolerance test, the blood glucose of group BL was maintained the lowest level (p<0.05), and the area under the blood glucose curve (AUC) was also significantly lower in group BL than in group DC (p<0.05). The fasting blood glucose and insulin levels after the experiment were significantly low in group BL (p<0.05), and the HOMA-IR was more significantly low in groups BL and EF than in group DC (p<0.05). Also, the HbA1c content of group BL was significantly low than in groups DC and RG (p<0.05). The serum TC level was significantly decreased in groups RG, BL, and EF than in group DC (p<0.05), and the LDL-C content was significantly low in group BL than in group DC (p<0.05). From the findings, it was shown that the puffed and fermented red ginseng made using a mixed strain culture of $B.$$breve$ and $L.$$delbrueckii$ could improve blood glucose-related biomarkers.
Carrot (Daucus carota L. var. sativa) is one of the most extensively used vegetable crops in the world and a significant source of nutrient because of its high content of ${\beta}$-carotene, well known as the precursor of vitamin A carotenoid. However, seed-hairs generated and elongated from the epidermal cell of seeds inhibit absorption and germination by various factors such as carotol and so on. Accordingly, mechanical hair removal process is essential before commercialization of carrot seeds. Because of this process, producers will have additional losses such as time consuming, manpower, capital and so on. Furthermore, physical damage of seeds causes irregular germination rate. To overcome such cumbersome weaknesses, new breeding program for developing hairless-seed carrot cultivar has been needed and studies for molecular markers related to seed-hair characteristic is needed for a new breeding program. Therefore, in this study, cDNA libraries from seeds of short-hair seed phenotype CT-SMR 616 OP 659-1 line, hairy-seed phenotype CT-SMR 616 OP 677-14 line and short-hair seed phenotype CT-ATR 615 OP 666-13 line, hairy-seed phenotype CT-ATR 615 OP 671-9 were constructed, respectively. Furthermore, 1,248 ESTs in each line, total 4,992 ESTs were sequenced. As a result, 19 SNP sites and 14 SNP sites in each of 2 combinations were confirmed by analyzing these EST sequences from short-hair and hairy-seed lines. Then we designed SNP primer sets from EST sequences of SNP sites for high resolution melting (HRM) analysis. Designed HRM primers were analyzed using hairy seed phenotype CT-SMR 616 OP 1040 line and short-hair seed phenotype CT-SMR 616 OP 1024, 1025, 1026 lines. One set of HRM primers showed specific difference between the melting curves of hairy and short-hair seed phenotype lines. Based on this result, allele-specific (AS) PCR primers were designed for easier selection between hairy-seed carrot and hairless seed carrot. These results of HRM and AS-PCR are expected to be useful in breeding of hairless seed carrot cultivar as a molecular marker.
The pharmacokinetic properties of amoxicillin trihydrate (Amox) were studied after single oral administration and single intravenous injection to cultured eel, Anguilla japonica, respectively (average $220{\pm}10\;g$, $28{\pm}1^{\circ}C$). Plasma samples were taken at 3, 5, 10, 15, 24, 30, 48, 96 and 144 h post-dose. The kinetic profile of absorption, distribution and elimination of Amox in plasma were analyzed fitting to a two-compartment model by WinNonlin program. In oral dosage of 40 and 80 mg/kg body weight, the peak plasma concentrations of Amox, which attained at 3~12 h post-dose, were 3.4 and $3.3\;{\mu}g/ml$, respectively. In intravenous injection with 1 mg/kg, the peak plasma concentrations of Amox, which attained at 9 h post-dose, was $7.2\;{\mu}g/ml$. The following parmeters were calculated for a single oral dosage of 40 and 80 mg/kg body weight, respectively: AUC (the area under the concentration-time curve)= 464 and $667\;{\mu}g{\cdot}h/ml$; $T_{max}$ (time for maximum concentration)= 2.1 and 3.6 h; $C_{max}$ (maximum concentration)= 3.04 and $3.4\;{\mu}g/ml$. Following intravenous injection at 1 mg/kg, this parameters were AUC= $748\;{\mu}g{\cdot}h/ml$; $C_{max}=4.2\;{\mu}g/ml$. The apparent oral bioavailability at 40 and 80 mg/kg were 1.6 and 1.1%, respectively. Despite using the trihydrate form of amoxicillin, the oral bioavailability was low in eel.
This study was conducted to investigate the changes of land use and stand volume around Mt. Kuem-O by B/W aerial photographs in 1979 and B/W Infrared aerial photographs in 1988. The results obtained in this study were as follow : 1. In classification of forest type on aerial photographs, coniferous stand was dark tone and hardwood stand was light tone and irregularly rounded crowns. 2. In classification of coniferous stand, Pinus densiflora was narraw cone and rounded tip of crowns and rough texture, Pinus rigida was irregulary rounded and broadly conical crowns. 3. To refer to changes of forest land area, mixed forest was changed into P. desiflora (687ha), P. rigida (130ha) and hardwood stand (219ha). 4. The regression equations between crown diameter and DBH were significant at 1% level by F-test in all stands. So the equation, D=a+bCD was used to estimate DBH. 5. The tree height curve equations were significant at 1% level by F-test in all stands. To estimate tree height the equation, logH=loga+blogD was adopted in P. densiflora and L. leptolepis and $H=a-bD+cD^2$ was adopted in P. rigida, hardwood stand and mixed forest. 6. The highest volume per hectare was observed in L. leptolepis and mixed forest showed the greatest growth percentage, while the lowest volume per hectare and growth percentage were observed in hardwood stand.
This study was conducted to estimate the volume growth by forest type and major species using the national forest resource inventory and to predict the final age of maturity by deriving the mean annual increment (MAI) and the current annual increment (CAI). We estimated the volume growth using the Chapman-Richards model. In the volume estimation equations by forest type, coniferous forests exhibited the highest growth. According to the estimation formula for each major species, Larix kaempferi will grow the highest among coniferous tree species and Quercus mongolica among broad-leaved tree species. And these estimation formulas showed that the fitness index was generally low, such as 0.32 for L. kaempferi and 0.21 for Quercus variabilis. In the analysis of residual amount, which indicates the applicability of the volume estimation formula, the estimates of the estimation formula tended to be underestimated in about 30 years or more, but most of the residuals were evenly distributed around zero. Therefore, these estimation formulas have no difficulty estimating the volume of actual forest species in Korea. The maximum age attained by calculating MAI was 34 years for P. densiflora, 35 years for L. kaempferi, and 31 years for P. rigida among coniferous tree species. In broad-leaved tree species, we discovered that the maximum age was 32 years for Q. variabilis, 30 years for Q. acutissima, and 29 years for Q. mongolica. We calculated MAI and CAI to detect the point at which these two curves intersected. This point was defined by the maximum volume harvesting age. These results revealed no significant difference between the current standard cutting age in public and private forests recommended by the Korea Forest Service, supporting the reliability of forestry policy data.
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