• Journal of Life Science
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• v.25 no.9
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• pp.1021-1026
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• 2015

Invertase (β-D-fructosfuranosidase, EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Three biochemical subgroups of invertases have been investigated in plants: vacuolar (soluble acid), cytoplasmic (soluble alkaline), and cell wall-bound (insoluble acid) invertases. An isoform of neutral invertase was purified from pea seedlings (Pisum sativum L.) and treated with gibberellic acid (GA) by sequential procedures consisting of ammonium sulfate precipitation, ion-exchange chromatography, absorption chromatography, and reactive green-19 affinity chromatography. The results of the overall insoluble invertase purification were a 430-fold increase. The purified neutral invertase was not glycosylated and had an optimum pH between neutral and alkaline (pH 6.8-7.5). It was inhibited by Tris, as well as by heavy metals, such as Hg²⁺ and Cu²⁺. Typical Michaelis–Menten kinetics were observed when the activity of the purified invertase was measured, with sucrose concentrations up to 100 mM. The K_m and V_max values were 12.95 mM and 2.98 U/min, respectively. The molecular mass was around 20 kDa. The sucrose-cleaving enzyme activity of this enzyme is similar to that of sucrose synthase and fructosyltransferase, but its biochemical characteristics are different from those of sucrose synthase and fructosyltransferase. Based on this biochemical characterization and existing knowledge, neutral INV is an invertase isoform in plants.

• Biomolecules & Therapeutics
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• v.24 no.4
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• pp.410-417
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• 2016

Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin-3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance ${\alpha}7$ nicotinic acetylcholine receptor (${\alpha}7$ nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of ${\alpha}7$ nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current ($I_{ACh}$) in Xenopus oocytes expressing the ${\alpha}7$ nAChR. $I_{ACh}$ was measured with a two-electrode voltage clamp technique. In oocytes injected with ${\alpha}7$ nAChR copy RNA, quercetin enhanced $I_{ACh}$, whereas quercetin glycosides inhibited $I_{ACh}$. Quercetin glycosides mediated an inhibition of $I_{ACh}$, which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of $I_{ACh}$ inhibition by quercetin glycosides was Rutin${\geq}$Rham1>Rham2. Quercetin glycosides-mediated $I_{ACh}$ enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated $I_{ACh}$ inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated ${\alpha}7$ nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the ${\alpha}7$ nAChR in a differential manner.

• Korean Journal of Clinical Laboratory Science
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• v.50 no.3
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• pp.297-303
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• 2018

This study investigated the effect of community-based health intervention program to improve metabolic disease in Jeju island. There were 50 obese local residents in the experimental group (body mass index, $BMI{\geq}25kg/m^2$). They participated in cooking therapy with nutrition education (4 times), exercise program with proprioceptive neuromuscular facilitation (PNF) and stretching training (9 times), and alternative medicine program with healing touch massage (3 times) during a 3-week period. To evaluate the effectiveness of the program, body composition, blood lipid profiles, blood glucose, and waist circumference were assessed before and after the intervention program. After the program, the value of total cholesterol (TC), glycosylated hemoglobin (HbA1C), diastolic blood pressure, percent body fat, and waist circumference were decreased, and became the normal value. Especially, HbA1C, percent body fat, and waist circumference were significantly decreased (P<0.001) in the experimental group, while HbA1C, percent body fat, and waist circumference were significantly increased (P<0.001) in the control group (N=50), who had no intervention. Our results suggest that metabolic syndrome associated markers need intervention program for improving them. In conclusion, although this study did not analyze the effect of the health intervention program and cooking therapy separately, considering the result of this 3-week, short term program, the effect will be more profuse if cooking therapy and exercise program were performed concurrently.

• Korean Journal of Food Science and Technology
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• v.39 no.1
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• pp.77-82
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• 2007

From the total methanolic extract of Chrysanthemum coronarium L. var. spatiosum (Compositae), nitrite scavenging ability and superoxide dismutase (SOD)-like activity were analyzed as antioxidative characteristics. After successive partitioning with chloroform, n-butanol, and water, the chloroform fraction showed the most significant nitrite scavenging ability with an $IC_{50}$ value of 39 ppm compared with the values of vitamin C and chlorogenic acid, 15 ppm and 36 ppm, respectively. The active fraction was subjected to silica gel and Sephadex LH-20 column chromatography, and the compound was isolated and identified as ${\beta}-sitosterol-O-{\beta}-D-glucoside$ using $^{1}H-NMR$ and $^{13}C-NMR$ spectral data. The glucoside was further hydrolyzed and confirmed as a glycosylated ${\beta}-sitosterol$. The compound and its aglycone, ${\beta}-sitosterol$, showed different nitrite scavenging and SOD-like activity. The $IC_{50}$ value of nitrite scavenging ability of the compound was 335 ppm at pH 1.5, while that of its aglycone was 41 ppm. As for the SOD-like activity, the $EC_{50}$ values of the sterol and the glucoside were 1,291 ppm and >2,000 ppm, respectively, compared with those of vitamin C and chlorogenic acid, 38 ppm and 449 ppm, respectively.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.406-412
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• 1998

The endoglucanase gene, egl6, of Trichoderma sp. was connected with the yeast ADH1 promoter, and the resultant plasmid, pVT-C4, was introduced into three S. cerevisiae host strains (YNN27, 2805, and SEY2102). Among each 80 transformants, the cell growth and expression level of endoglucanase were compared in test-tube cultivation, and three respective transformants for each host cells showing the highest expression level and cell growth were selected. When three recombinant yeast cells were batchwise cultivated for 48 hr in flask, the total activities of endoglucanase expressed were about 1140 unit/l with 2805/pVT-C4, 1020 unit/l with SEY2102/pVT-C4, and 590 unit/l with YNN27/pVT-C4. Irrespective of host strain, about 80% of the expressed endoglucanase was detected in the extracellular medium. In addition, it was also found that the recombinant enzyme was secreted into the culture medium as two major forms of lightly and heavily glycosylated proteins.

• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1547-1553
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• 2018

Hyaluronidases are a family of enzymes that catalyse the breakdown of hyaluronic acid, which is abundant in the extracellular matrix and cumulus oocyte complex. To investigate the activity of recombinant bovine sperm hyaluronidase 1 (SPAM1) and determine the effect of the Asn-X-Ser/Thr motif on its activity, the bovine SPAM1 open reading frame was cloned into the mammalian expression vector pCXN2 and then transfected to the HEK293 cell line. Expression of recombinant bovine hyaluronidase was estimated using a hyaluronidase activity assay with gel electrophoresis. Recombinant hyaluronidase could resolve highly polymeric hyaluronic acid and also caused dispersal of the cumulus cell layer. Comparative analysis with respect to enzyme activity was carried out for the glycosylated and deglycosylated bovine sperm hyaluronidase by N-glycosidase F treatment. Finally, mutagenesis analysis revealed that among the five potential N-linked glycosylation sites, only three contributed to significant inhibition of hyaluronic activity. Recombinant bovine SPAM1 has hyaluronan degradation and cumulus oocyte complex dispersion ability, and the N-linked oligosaccharides are important for enzyme activity, providing a foundation for the commercialization of hyaluronidase.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• International Journal of Industrial Entomology and Biomaterials
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• v.36 no.1
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• pp.15-24
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• 2018

N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (${\alpha}1AGP$) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the ${\alpha}1AGP$ to be a better model for studying glycosylation. The modified ${\alpha}1AGP$ (${\alpha}1AGP{\Delta}$) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the ${\alpha}1AGP$. Subsequently, we confirmed the detailed profile of N-glycan on the ${\alpha}1AGP{\Delta}$ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant ${\alpha}1AGP{\Delta}$ could be usable as a better model glycoprotein of N-glycosylation research in BES.

• Korean Journal of Medicinal Crop Science
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• v.10 no.4
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• pp.253-258
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• 2002

We compared the long-term metabolic effects of equal amounts of carbohydrate from potato, rice and buckwheat on glycemic indices and blood lipids in healthy subjects. Nine healthy volunteers-2 men and 7 women were studied. All subjects ate diets based on the same-7-day rotating menu differing only in that the major source of carbohydrate (about 50% of daily total calories) came either from buckwheat, rice or potato. The study was conducted with a triple crossover design over three 7 day periods. On the morning of the 8th day, fasting blood was drawn from each subject to determine serum glucose, insulin, triglycerides, total and HDL-cholesterol. Subjects were then asked to eat breakfast with their respective carbohydrate within a 20 min period. Blood samples were drawn at 30, 60, 120 and 180 min after the start of breakfast to determine glucose and insulin levels. At 30 min the glucose response to the rice meal(7.15mmol/L) and potato meal(6.71mmol/L) were greater than the response to the buckwheat meal(5.855mmol/L) (P < 0.05). The mean area under the glucose response to the curve following the rice meal was greater than that following the buckwheat meal(P < 0.05). The insulin responses to the potato and rice meals at 30 and 60 min were greater than those to the buckwheat meal (P < 0.05). The mean area under the serum insulin response curve after the rice meal was greater than of buckwheat. Blood lipids, uric acid and glycosylated hemoglobin were not affected by the three meals. The study shows that the buckwheat meal has more beneficial effects on glycemic indices than either the rice meal or potato meal in healthy subjects.

• Journal of dental hygiene science
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• v.14 no.2
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• pp.107-113
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• 2014

This study was conducted to evaluate the association between periodontal disease and diabetes mellitus. The final analysis population of this study was composed of 4,830 adults with diabetes mellitus or periodontal disease and aged 19 years or older, based on the third-edition data of the 4th Korea National Health and Nutrition Examination Survey (KNHANES) (in 2009). Diabetic status and potential confounders were used in questionnaire materials and physical examination materials, and the presence of periodontal disease was used in the materials for oral health examination by a dentist. For diabetic status, fasting plasma glucose (FPG) levels <100 mg/dl were subcategorized into normal group and FPG levels ${\geq}100mg/dl$ into impaired fasting glucose group; glycosylated hemoglobin (HbA1c) levels <7% into normal group and HbA1c ${\geq}7%$ into diabetes group, on the basis of the American Diabetes Association. According to the 2009 Korea Health Statistics, the case where the pocket depth is 3 mm or more was defined as periodontal disease. The association between the two diseases was evaluated through $x^2$-test and logistic regression analysis using R-commander 2.14. In impaired fasting glucose group, community periodontal index (CPI) groups 3 to 4 had higher risks for periodontal disease 1.23 times (95% confidence interval, 1.07~1.42) than those of CPI groups CPI 0~2, even after adjustment for several confounders. In addition, periodontal disease and diabetes mellitus showed statistically significant differences depending on age, sex, income level, educational background, smoking status, alcohol consumption, and snack intake. The analyses of the third-edition data of the 4th KNHANES showed that there was a statistically significant association between periodontal disease and diabetes mellitus as examined by means of CPI in this study.