• Journal of Microbiology and Biotechnology
• /
• 제27권2호
• /
• pp.372-379
• /
• 2017

The transport of lysosomal enzymes into the lysosomes depends on the phosphorylation of their chains and the binding of the phosphorylated residues to mannose-6-phosphate receptors. The efficiency of separation depends more on the phosphodiesterases (PDEs) than on the activity of the phosphorylation of mannose residues and can be determined in vitro. PDEs play important roles in regulation of the activation of lysosomes. The expression of proteins was confirmed by western blotting. All PDE4 series protein expression was reduced in high concentrations of rolipram. As a result of observing the fluorescence intensity after rolipram treatment, the lysosomal enzyme was activated at low concentrations and suppressed at high concentrations. High concentrations of rolipram recovered the original function. Antimicrobial activity was not shown in either 10 or $100{\mu}M$ concentrations of rolipram in treated HeLa cells in vitro. However, the higher anticancer activity at lower rolipram concentration was shown in lysosomal enzyme treated with $10{\mu}M$ of rolipram. The anticancer activity was confirmed through cathepsin B and D assay. Tranfection allowed examination of the relationship between PDE4 and lysosomal activity in more detail. Protein expression was confirmed to be reduced. Fluorescence intensity showed decreased activity of lysosomes and ROS in cells transfected with the antisense sequences of PDE4 A, B, C, and D. PDE4A showed anticancer activity, whereas lysosome from cells transfected with the antisense sequences of PDE4 B, C, and D had decreased anticancer activity. These results showed the PDE4 A, B, C, and D are conjunctly related with lysosomal activity.

• Bulletin of the Korean Chemical Society
• /
• 제25권8호
• /
• pp.1195-1201
• /
• 2004

Tyrosinase was covalently immobilized on platinum electrode according to the method we developed for laccase (Bull. Korean Chem. Soc. 2002, 23(7), 385) and p-chlorophenol, p-cresol, and phenol could be detected with sensitivities of 334, 139 and 122 nA/ ${\mu}M$ and the detection limits of 1.0, 2.0, and 2.5 ${\mu}M$, respectively. The response time ($t_{90\%}$) is 3 seconds for p-chlorophenol, and 5 seconds for p-cresol and phenol. The optimal pHs of the sensor are in the range of 5.0- 6.0. This sensor can tolerate at least 500 times repeated injections of p-chlorophenol with retaining 80% of initial activity. In case of tyrosinase and laccase co immobilized platinum electrode, the sensitivities are 560 nA/ ${\mu}M$ for p-phenylenediamine (PPD) and 195 nA/ ${\mu}M$ for p-chlorophenol, respectively. The sensitivity of the bi-enzyme sensor for PPD increases 70% compared to that of only laccase immobilized one, but the sensitivity for p-chlorophenol decreases 40% compared to that of only tyrosinase immobilized one. The sensitivity increase for the bi-enzyme sensor for PPD can be ascribed to the additional catalytic function of the co-immobilized tyrosinase. The sensitivity decrease for p-chlorophenol can be explained by the “blocking effect” of the co-immobilized laccase, which hinders the mass transport through the immobilized layer. If PPD was detected with the electrode that had been used for p-chlorophenol, the sensitivity decreased 20% compared to that of the electrode that had been used only for PPD. Similarly, if p-chlorophenol was detected with PPD detected electrode, the sensitivity also decreased 20%. The substrate-induced conformation changes of the enzymes in a confined layer may be responsible for the phenomena.

• Molecules and Cells
• /
• 제28권5호
• /
• pp.479-487
• /
• 2009

Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semi-quantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to $H_2O_2$, menadione, and heavy metal ($CdCl_2$, $ZnCl_2$ and $AlCl_2$)-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to $H_2O_2$ stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.

• Clinical and Experimental Pediatrics
• /
• 제62권8호
• /
• pp.307-311
• /
• 2019

Purpose: Necrotizing enterocolitis (NEC) is one of the most serious complications of prematurity. Many risk factors can contribute to the development of NEC. The epidermal growth factor (EGF) plays a major role in intestinal barrier function, increases intestinal enzyme activity, and improves nutrient transport. The aim of this study was to assess the role of epidermal growth factor in the development of NEC in preterm neonates. Methods: In this study, 130 preterm neonates were included and divided into 3 groups, as follows: group 1, 40 preterm neonates with NEC; group 2, 50 preterm neonates with sepsis; and group 3, 40 healthy preterm neonates as controls. The NEC group was then subdivided into medical and surgical NEC subgroups. The serum EGF level was measured using enzyme-linked immunosorbent assay. Results: Serum EGF levels (pg/dL) were significantly lower in the NEC group (median [interquartile range, IQR], 9.6 [2-14]) than in the sepsis (10.1 [8-14]) and control groups (11.2 [8-14], P<0.001), with no significant difference between the sepsis and control groups, and were positively correlated with gestational age (r=0.7, P<0.001). A binary logistic regression test revealed that low EGF levels and gestational ages could significantly predict the development of NEC. The receiver-operating characteristic curve for EGF showed an optimal cutoff value of 8 pg/mL, with 73.3% sensitivity, 98% specificity, and an area under the curve of 0.92. Conclusion: The patients with NEC in this study had significantly lower serum EGF levels (P<0.001), which indicated that EGF could be a reliable marker of NEC in preterm neonates.

• Reproductive and Developmental Biology
• /
• 제39권4호
• /
• pp.97-104
• /
• 2015

Glucose is universal and essential fuel of energy metabolism and in the synthesis pathways of all mammalian cells. Glucose is the one of the major precursors of lactose synthesis using glycolysis result in producing milk fat and protein. During the milk fat synthesis, lipoprotein lipase (LPL) and CD36 are required for glucose uptake. Various morecules such as acyl-CoA synthetase 1 (ACSL1) activity of acetyl-CoA synthetase 2 (ACSS2), ACACA, FASN AGPAT6, GPAM, LPIN1 are closely related with milk fat synthesis. Additionally, glucose plays a major role for synthesizing lactose. Activations of lactose synthesize enzymes such as membranebound enzyme, beta-1,4-galactosyl transferase (B4GALT), glucose-6-phosphate dehydrogenase (G6PD) are changed by concentration of glucose in blood resulting change of amount of lactose production. Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose over a plasma membrane. There are 2 types of glucose transporters which consisted facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Among them, GLUT1, GLUT8, GLUT12, SGLT1, SGLT2 are main glucose transporters which involved in mammary gland development and milk synthesis. However, more studies are required for revealing clear mechanism and function of other unknown genes and transporters. Therefore, understanding of the mechanisms of glucose usage and its regulation in mammary gland is very essential for enhancing the glucose utilization in the mammary gland and improving dairy productivity and efficiency.

• 한국지하수토양환경학회:학술대회논문집
• /
• 한국지하수토양환경학회 2006년도 총회 및 춘계학술발표회
• /
• pp.54-56
• /
• 2006

Single-well-gas-sparging tests were developed and evaluated for assessing the feasibility of in-situ aerobic cometabolism of trichloroethylene (TCE), using propane as a growth substrate. To evaluate transport characteristics of dissolved solutes [sulfur hexafluoride (SF6) or bromide (non-reactive tracers), propane (a growth substrate), ethylene, propylene (nontoxic surrogates to probe for CAH transformation activity), and DO], push-pull transport tests were performed. Mass balance showed about 90% of the injected bromide and about 80% of the injected SF6 were recovered, and the recoveries of other solutes were comparable with bromide and slightly higher than SF6. A series of Gas-Sparging Biostimulation tests were performed by sparging propane/oxygen/argon/SF6 gas mixtures, and temporal ground water samples were obtained from the injection well under natural gradient 'drift' conditions. The decreased time for propane depletion and the longer time to deplete SF6 as a conservative tracer indicate the progress of biostimulation. Gas-Sparging Activity tests were performed. .Propane utilization, DO consumption, and ethylene and propylene cometabolism were well demonstrated. The stimulated propane-utilizers cometabolized ethylene and propylene to produce ethylene oxide and propylene oxide, as cometabolic by-products, respectively. Gas-Sparging Acetylene Blocking tests were performed by sparging gas mixtures including acetylene to demonstrate the involvement of monooxygenase enzymes. Gas substrate degradation was essentially completely Inhibited in the presence of acetylene, and no production of the corresponding oxides was also observed. The Gas-Sparging tests supports the evidences that the successive stimulation of propane-oxidizing microorganisms, cometabolic transformation of ethylene and propylene by the enzyme responsible for methane and propane degradation.

• 한국식품과학회지
• /
• 제12권1호
• /
• pp.45-52
• /
• 1980

E. coli로부터 분리 정제한 ${\beta}-galactosidase$를 1,6-diaminohexane과 sebacoyl chloride를 사용하여 계면증합 반응에 의하여 나일론 막에 microencapsulation 시켜 고정화 시켰다. 얻어진 microcapsule은 구형이었고 평균 직경이 $80{\mu}$이었으며 이 방법에 의하여 microencapsulation 시킨 ${\beta}-galactosidase$의 효소 역가 효율은 45%이었다. 유당의 막 투과는 거의 완전하게 이루어졌다. Microencapsulation 시킨 ${\beta}-galactosidase$의 성질은 가용성 효소와 거의 비슷하였고 최적 pH는 $7.0{\sim}7.2$에서 $7.3{\sim}7.5$로 약간 이동하였으며, 최적 온도는 $50^{\circ}C,\;K_m$값은 o-nitrophenyl-${\beta}$-D-galactopyranoside(ONPG)와 유당에 대하여 가용성 효소는 각각 $3.33{\times}10^{-4},$ 및 $2.86{\times}10^{-3}M$ 이었고 고정화 효소는 $5.28{\times}10^{-4}$ 및 $4.25{\times}10^{-3}M$이었다. 활성화 에너지는 가용성 효소는 8.94와 고정화 효소는 9.78 Kcal/mole이었다. 이 고정화 효소를 사용하여 5% 표준 유당 용액과 탈지 우유에 존재하는 유당을 가수 분해한 결과 40시간안에 각각 80 및 70%씩 가수 분해 하였다. 또한 공정중의 고정화 효소의 안정성을 살펴 본 결과 $27^{\circ}C$에서 한번에 24시간씩 5번 사용 후 남아 있는 역가는 50%로서 실제 이용상 긍정적인 결과를 나타내었다.

• 대한간호학회지
• /
• 제13권2호
• /
• pp.87-104
• /
• 1983

It has been well documented that animals exposed to cold show increased activity of thyroid gland. The calorigenic action of thyroid hormone has been demonstrated by a variety of in vivo and in vitro studies. According to Edelman et al., the thyroid thermogenesis is due to activation of energy consuming processes, especially the active sodium transport by the hormone in target tissues. If so, the increase in thyroid activity during cold exposure should induce increased capacity of sodium transport in target tissue and the change in tissue metabolism should be precisely correlated with the change in Na+_K+_ATPase activity of the tissue. This possibility was tested in the present study: in one series, changes in oxygen consumption and Na+_K+_-ATPase activity of liver preparations were measured in rats as a function of thyroid status, in order to establish the effect of thyroid hormone on the tissue respiration and enzyme system in another series, the effect of cold stimulus on the serum thyroid hormone level, hepatic tissue oxygen consumption and Na+_K+_ATPase activity in rats. The results obtained are as follows: 1. The Na+_dependent oxygen consumption of liver slices, the oxygen consumption of liver mitochondria and the Na+_K+_ATPase activity of liver preparations were significantly inhibited in hypothyroidism and activated in hyperthyroidism. Kinetic analysis indicated that the Vmax. of Na+_K+_ATPase was decreased in hypothyroidism and increased in hyperth)'roidism. 2. In cold exposed rats, the serum triiodothyronine (T₃) level increased rapidly during the initial one day of cold exposure, then declined slowly to the control level after two weeks. The serum thyroxine (T₄) level decreased gradually throughout the cold exposure. Accordingly the T₃/T₄ratio increased. The mitochondrial oxygen consumption and the Na+_dependent oxygen consumption of liver slices increased during the first two days and then remained unchanged thereafter The activity of the Na+_K+_ATPase in liver preparations increased during cold exposure with a time course similar to that of oxygen consumption. Kinetic analysis indicated that the Vmax. of Na+_K+_ATPase increased. 3. Once the animal was adapted to cold, induction of hypothyroidism did not significantly alter the hepatic oxygen consumption and Na+_K+_ATPase activity. These results indicate that: 1) thyroid hormone increases capacities of mitochondrial respiration and active sodium transport in target tissues such as liver; 2) the increased T₃level during the initial period of cold exposure facilitates biosynthesis of Na+_K+_ATPase and mitochondrial enzymes for oxidative phosphorylation, leading to enhanced production and utilization of ATP, hence heat production.

• BMB Reports
• /
• 제36권1호
• /
• pp.110-119
• /
• 2003

The acetylation state of histone is reversibly regulated by histone acetyltransferase (HAT) and deacetylase (HDAC). An imbalance of this reaction leads to an aberrant behavior of the cells in morphology, cell cycle, differentiation, and carcinogenesis. Recently, these key enzymes in the gene expression were cloned. They revealed a broad use of this modification, not only in histone, but also other proteins that involved transcription, nuclear transport, and cytoskeleton. These results suggest that HAT/HDAC takes charge of multiple-functions in the cell, not just the gene expression. HDAC is especially known to play an important role in carcinogenesis. The enzyme has been considered a target molecule for cancer therapy. The inhibition of HDAC activity by a specific inhibitor induces growth arrest, differentiation, and apoptosis of transformed or several cancer cells. Some of these inhibitors are in a clinical trial at phase I or phase II. The discovery and development of specific HDAC inhibitors are helpful for cancer therapy, and decipher the molecular mode of action for HDAC.

• 한국토양비료학회지
• /
• 제44권1호
• /
• pp.84-90
• /
• 2011

It is imperative to study the hydrolysis of urea in high saline-sodic condition of a newly reclaimed tidal land in order to overcome the problems associated with use of urea fertilizer. The methodology adopted in this study tried to get a convenient way of estimating rate for N transformation needed in N fate and transport studies by reviewing pH and salt contents which can affect the microbial activity which is closely related to the rate of urea hydrolysis. The hydrolysis of urea over time follows first-order kinetics and soil urease activity in reclaimed soils will be represented by Michaelis-Menten-type kinetics. However, high pH and less microorganisms may delay the hydrolysis of urea due to decrease in urease activity with increasing pH. Therefore, the rate of urea hydrolysis should adopt $V_{max}$ referring enzyme activity ($E_0$) accounting for urease concentration which is indicative for urea hydrolysis, especially in a high saline and sodic soils.