• Biotechnology and Bioprocess Engineering:BBE
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• 제3권1호
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• pp.24-31
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• 1998

The separation and concentration of L-phenylalanine (L-Phe) using a supported liquid membrane (SLM) is investigated. A cation complex agent, di-2-ethylhexyl phosphoric acid (D2EHPA), is used as a carrier in the SLM with n-Heptane as a solvent. The reaction order and equilibrium constant in the formation reaction of L-phe-carrier complex are obtained from the extraction experiment. A mathematical model for a carrier mediated counter transport process is proposed to estimate the diffusion coefficient of L-phe-carrier complexly in the liquid membrant. Permeation experiments of L-phe using a SLM are performed under various operating conditions and optimum conditions for the transport of L-phe are obtained. Concentration of L-phe in the strip phase against its concentration is observed. Transport rate of glucose through liquid membrane is less than that of L-phe in the competitive transport of L-phe and glucose. And the existence of glucose reduced the transport rate of L-phe. The performance of separation with continuous strip phase is increased due to the dilution effect in the strip phase.

• 한국미생물·생명공학회지
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• 제19권2호
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• pp.128-134
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• 1991

To study the reduced growth and synthesis, proeviously reported, of ${\beta}$-galactosidase of alkalophilic Bacillus sp. YS-309 at the higher lactose concentration of 0.5% (w/v) in the medium, lactose transport and utilization were examined. The results showed that lactose transport was influenced by the addition of four kinds of antibiotics, and tetracycline stimulated most but not valinomycin. PEP-potentials of the cells grown on lactose was estimated lower than the cells on glucose and on galactose. Thus, the transport of lactose was independent of intracellular PEP and phosphorylation reactions, and was thought to be uptaked directly or oxidized in part in the transport process. In the other hand, once lactose was uptaked into the cells, it was hydrolyzed by ${\beta}$-glactosidase to glucose and galactose. The former was metabolized fast but the latter was accumulated. Galactose and lactose were not utilized until glucose was mostly depleted in the medium. The ${\beta}$-galactosidase synthesis decreased in the presence of glucose over 0.2% and galactose over 0.05 to 0.1%, respectively. In conclusion, it was considered for glucose as a repressor and galactose as a inducer for ${\beta}$-galactosidase synthesis even though the mechanisms were not elucidated. Catabolite repression of glucose on the enzyme synthesis was not relieved by the addition of exogeneous cAMP.

• The Korean Journal of Physiology and Pharmacology
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• 제4권2호
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• pp.91-97
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• 2000

The purpose of the present study was to determine the preventive effects of combined interventional trial of fish oil treatment and exercise training on insulin resistance of skeletal muscle in high-fat fed rats. Male Wistar rats were randomly divided into chow diet (CD), high-fat diet (HF), high-fat diet with fish oil (FO), high-fat diet with exercise training (EX), and FO+EX groups. The rats in control group were fed chow diet containing, as percents of calories, 58.9% carbohydrate, 12.4% fat, and 28.7% protein. High-fat diet provided 32% energy as lard, 18% as corn oil, 27% as carbohydrate and 23% as casein. The fish oil diet had the same composition as the high fat diet except that 100 g menhaden oil was substituted for corn oil. Insulin sensitivity was assessed by in vitro glucose transport in the soleus muscle after diet treatment and treadmill running for 4 weeks. While the FO or EX only partially prevented insulin resistance on glucose transport and visceral obesity induced by high-fat diet, these interventions completely corrected hyperinsulinemia and hyperglycemia from the high-fat diet. The rats in the FO+EX showed normalized insulin action on glucose transport, plasma chemicals and visceral fat mass. Insulin-mediated glucose transport was negatively associated with total visceral fat mass (r=－0.734; p＜0.000), plasma triglyceride (r=－0.403; p＜0.05) and lepin (r=－0.583; p＜0.001) concentrations with significance. Multiple stepwise regression analysis showed that only total visceral fat mass was independently associated with insulin-mediated glucose transport (r=－0.668; p＜0.000). In conclusion, combined interventional trial of FO+EX recovered insulin resistance on glucose transport of skeletal muscle induced by high-fat diet. Visceral fat mass might be more important factor than plasma TG and leptin to induce insulin resistance on glucose transport of skeletal muscle in high-fat fed rats.

• 대한의생명과학회지
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• 제9권4호
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• pp.177-181
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• 2003

The baculovirus/Spodoptera frugiperda (Sf) cell system has become popular for the production of large amounts of the human erythrocyte glucose transporter, GLUT1, heterologously. However, it was not possible to show that the expressed transporter in insect cells could actually transport glucose. The possible reason for this was that the activity of the endogenous insect glucose transporter was extremely high and so rendered transport activity resulting from the expression of exogenous transporter very difficult to detect. Sf21-AE cells are commonly employed as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains 0.1 % D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike the human glucose transporter, very little is known about properties of the endogenous sugar transporter(s) in insect cells. Thus, the uptake of 2-deoxy-D-glucose (2dGlc) by Sf21-AE cells and the inhibition of 2dGlc transport in the insect cells by fructose and cytochalasin B were investigated in the present work. The binding assay of cytochalasin B was also performed, which could be used as a functional assay for the endogenous glucose transporter(s) in the insect cells. Sf21-AE cells were infected with the recombinant virus AcNPV-GT or no virus, at a multiplicity of infection (MOI) of 5. Infected cells were resuspended in PBS plus and minus 300 mM fructose, and plus and minus 20 $\mu$M cytochalasin B for use in transport assays. Uptake was measured at 28$^{\circ}C$ for 1 min, with final concentration of 1 mM deoxy-D-glucose, 2-[1,2-$^3$H]- or glucose, L-[l,$^3$H]-, used at a specific radioactivity of 4 Ci/mol. The results obtained demonstrated that the sugar uptake in uninfected cells was stereospecific, and was strongly inhibited by fructose but only poorly inhibitable by cytochalasin B. It is therefore suggested that the Sf21-AE glucose transporter has very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• 대한의생명과학회지
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• 제14권2호
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• pp.119-122
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• 2008

The baculovirus/insect cell expression system is of great value in the study of structure-function relationships in mammalian glucose-transport proteins by site-directed mutagenesis and for the large-scale production of these proteins for mechanistic and biochemical studies. In order to exploit this, the effects of substitution at the highly conserved residue glutamine 282 of the human erythrocyte-type glucose transporter have been examined by in vitro site-directed mutagenesis. The modified human transport protein has been expressed in Spodoptera frugiperda 21 cells by using the recombinant baculovirus AcNPV-GTL. To assess the functional integrity of the expressed transporter, measurements of the transport inhibitor cytochalasin B binding were performed, involving the membranes prepared from 4 days post infection with no virus, with wild-type virus or AcNPV-GTL virus. Data obtained showed that there was little or no D-glucose-inhibitable binding in cells infected with the wild type or no virus. Only the recombinant virus infected cells exhibited specific binding, which is inhibitable by D- but not by L-glucose. However, there was a notable reduction in the affinity for the potent inhibitor cytochalasin B when binding measurements of AcNPV-GTL were compared with those of AcNPV-GT, which has no substitution. It is thus suggested that although the modified and unmodified human transporters differed slightly in their affinity for cytochalasin B, the glutamine substitution did not interfere the heterologous expression of the human transporter in the insect cells.

• The Korean Journal of Physiology and Pharmacology
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• 제9권2호
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• pp.125-130
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• 2005

We examined the effect of leptin on the insulin resistance in skeletal muscles by measuring the glucose transport. Male Wistar rats were fed with chow or high-fat diets for 30 days. Three days before sacrifice, high-fat fed rats were subcutaneously injected with leptin (1 mg/kg body weight) for 3 days. The glucose transports in the epitrochlearis and soleus muscle were not different among the experimental groups under basal state, however these were decreased significantly in the high fat-diet rats under insulin-stimulation (p＜0.01). Leptin treatment recovered the decreased glucose transport in the epitrochlearis (p＜0.05) and soleus (p=0.08). Triglyceride concentration in the soleus muscle was increased significantly in the high fat-fed rats, compared to chow diet rats (p＜0.01), and it was decreased significantly by leptin treatment (p＜0.01). The glucose transport was measured under basal and $60{\mu}u/ml$ of insulin with or without 50 ng/ml of leptin. Leptin had no direct stimulatory effect on glucose transport under both basal and insulin-stimulated conditions in vitro. These results demonstrate that leptin injection to high fat diet fed rats recovered impaired insulin responsiveness of the skeletal muscles and muscle triglyceride concentration. However, there was no direct stimulatory effect of leptin on insulin sensitivity of the skeletal muscle in vitro.

• 대한의생명과학회지
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• 제11권3호
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• pp.327-332
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• 2005

Sf21 cells become popular as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike human glucose transporters, very little is known about the characteristics of the endogenoussugar transporter(s) in Sf21 cells. Thus, some kinetic properties of the sugar transport system were investigated, involving the uptake of 2-deoxy-D-glucose (2dG1c). In order to obtain a true measure of the initial rate of uptake, the uptake of $[^3H]2dGlc$ from both low $(100{\mu}M)$ and high (10 mM) extracellular concentrations was measured over periods ranging from 30 sec to30 min. The data obtained indicated that the uptake was linear for at least 2 min at both concentrations, suggesting that measurements made over a 1min time course would reflect initial rates of the jexpse uptake. To determine $K_m\;and\;V_{max}$ of the endogenous glucose transporter(s) in Sf21 cells, the uptake of 2dG1c was measured over a range of substrate concentrations $(50{\mu}M\~10mM)$ 2dG1c uptake by the Sf21 cells appeared to involve both saturable and non-saturable (or very low affinity) components. A saturable transport system for 2dG1c was relatively high, the $K_m$ value for uptake being < 0.45 mM. The $V_{max}$ value obtained for 2dG1c transport in the Sf21 cells was about 9.7-folds higher than that reported for Chinese hamster ovary cells, which contain a GLUT1 homologue. Thus, it appeared that the transport activity of the Sf21 cells was very high. In addition, the Sf21 glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter

• Environmental Analysis Health and Toxicology
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• 제20권1호
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• pp.75-85
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• 2005

This study was aimed to know the effect of cadmium chloride (CdCl₂) on glucose transport in L6 myotube and its action mechanism. CdCl₂ increased the 2-deoxy- (l-3H)-D-glucose (2-DOG) uptake 1.9 and 2.4 fold at 10 and 25 μM respectively. To investigate the stimulating-mechanism of glucose transport induced by CdCl₂, the wortmannin and PD98059 were used as PI3K (phosphatidylinositol 3-kinase) inhibitor and MAPK inhibitor respectively, which did not affect 2-DOG uptake. This fact suggests that CdCl₂ induced 2-DOG uptake may not be concerned to the insulin signalling pathway. Whereas nifedipine, a calcium channel blocker, and trifluoperazine, a calmodulin inhibitor, were found to inhibit the 2-DOG uptake stimulted by CdCl₂. In addition, we also measured the ROS (reactive oxygen species) production and GSH level in L6 myotube to investigate the correlation between the glucose uptake and ROS. CdCl₂(25 μM) increased ROS generation approximately 1.5 fold and changed the cellular GSH level, but GSSG/GSH ratio remained unchanged. CdCl₂ stimulated 2-DOG uptake and ROS generation were inhibited by N-acetylcystein. And BSO pretreatment, a potent inhibitor of γ-GCS, resulted in the dramatic decrease of 2-DOG uptake and also the increase of the sensitivity to cadmium cytotoxicity. The obtained results suggest that CdCl₂-stimulated glucose uptake might be based on the activation of HMP shunt as an antioxidant defense mechanism of the cells.

• 대한전기학회:학술대회논문집
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• 대한전기학회 1999년도 추계학술대회 논문집 학회본부 C
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• pp.984-986
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• 1999

In the case of immobilizing of glucose oxidase in organic polymer using electrosynthesis, the glucose oxidase obstructs charge transfer and mass transport during the film growth. This may lead to short chained polymer and make charge-coupling weak between the glucose oxidase and the backbone of the polymer. That is mainly due to insulating property and net chain of the glucose oxidase. Such being the case, it is useless to increase in amount of glucose oxidase more than reasonable in the synthetic solution. We establish by means of qualitative analysis that amount of immobilized glucose oxidase can be improved by adding a hole ethyl alcohol in the synthetic solution. As ethyl alcohol was added by 0.1mol $dm^{-3}$ in the synthetic solution, the faradic impedance of resultant electrode was increased about five times as much as the case of ethyl alcohol free in the solution, and mass transport was limited more than over. That is due to insulating property and net chain of the glucose oxidase. Moreover, in ultraviolet spectra of the synthetic solution, the adsorption peak at 285nm corresponding to glucose oxidase was decreased. It suggests increase in amount of immobilized glucose oxidase.

• Journal of Nutrition and Health
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• 제31권4호
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• pp.809-822
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• 1998

Retarding effects of the dietary fibers from persimmon peels (PTDF ; total dietary fibers, PIDF ; insoluble dietary fibers, PSDF ; soluble dietary fibers) and dried jujubes (JTDF ; total dietary fibers, JIDF ; insoluble dietary fibers, JSDF ; soluble dietary fibers) on glucose, bile acid and cadmium transport were evaluated by in vitro dialysis sack method. These effects were compared with those of commerical citrus pectin, CM-cellulose (CMC) and $\alpha$-cellulose. Yields of PTDF, PIDF and PSDF on wet weight basis were 14.04% , 11.61%, 1.76%, respectively, and those from dried jujubes were 14.80%, 9.98% and 2.80%, respectively. The amount of soluble fibers in JTDF was higher than PTDF. Soluble fibers had the retarding effects on glucose transport but insoluble fibers did not have. CM-cellulose showed the greatest retarding effect, which was followed by citrus pectin and JSDF. Among the soluble fibers, PSDF had the lowest retardig effect. Retarding effect of TDF was dependent upon the amount of SDF in TDF. Regarding bile acid dialysis , insoluble dietary fibers as well as soluble dietary fibers showed the retarding effects, among which JSDF had the greatest retarding effects, among which JSDF had the greatest retarding effect, followed by citrus pectin. Among the extracted fibers, dietary fibers from dried jujubes were more effective than these from persimmon peels, and SDF seemed to show higher retarding effects than IDF and TDF. On cadmium transport retardation , all dietary fibers except $\alpha$-cellulose had the retarding effects and PSDF showed the greatest effect which was followed by PIDF and CMC, The extracted fibers showed higher retarding effect on Cd transport than glucose and bile acid transport, and dietary fibers from persimmon peels showed higher retarding effects than those from dried jujubes.