• Applied Microscopy
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• v.32 no.2
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• pp.91-105
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• 2002

Urea transport in the kidney is mediated by a family of transporter proteins that includes renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). The cDNA of five isoforms of rat UT-A, UTA1, UT-A2, UT-A3, UT-A4, and UT-A5 have been cloned. The purpose of this study was to examine the expression of UT-A (L194), which marked UT-A1, UT-A2 and UT-A4. Male Sprague-Dawley rats, weighing approximately 200 g, were divided into three group: control rats had free access to water, dehydrated rats were deprived of water for 3 d, and water loaded rats had free access to 3% sucrose water for 3 d before being killed. The kidneys were preserved by in vivo perfusion through the abdominal aorta with the 2% paraformaldehyde-lysine- periodate (PLP) or 8% paraformaldehyde solution for 10 min. The sections were processed for immunohistochemical studies using pre-embedding immunoperoxidase method and immunogold method. In the normal rat kidney, UT-A1 was expressed intensely in the cytoplasm of the inner medullary collecting duct (IMCD) cell and UT-A2 was expressed on the plasma membrane of the terminal portion of the shortloop descending thin limb (DTL) cells (type I epithelium) and of the long-loop DTL cells (type II epithelium) in the initial part of the inner medulla. Immunoreactivity for UT-A1 in the IMCD cells, was decreased in dehydrated animals whereas strongly increased in water loaded animals compared with control animals. In the short-loop DTL, immunoreactivity for UT-A2 was increased in intensity in both dehydrated and water loaded groups. However, in the long-loop DTL of the outer part of the inner medulla, immunoreactivity for UT-A2 was markedly increase in intensity in dehydrated group, but not in water loaded group. In conclusion, in the rat kidney, UT-A1 is located in the cytoplasm of IMCD cells, whereas UT-A2 is located in the plasma membrane of both the short-and long-loop DTL cells. Immunohistochemistry studies revealed that UT-A1 and UT-A2 may have a different role in urea transport and are regulated by different mechanisms.

• Journal of Life Science
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• v.21 no.1
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• pp.119-126
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• 2011

There is a growing recognition of the significance of $H_2S$ as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine ${\beta}$-synthase (CBS) and cystathionine ${\gamma}$-lyase (CGL), are believed to be chiefly responsible for $H_2S$ biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down's syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for ${\alpha}-{\gamma}$ elimination reaction with L-cystathionine and 88% for ${\alpha}-{\beta}$ elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 nm absorption maximum, which is attributed to the binding cofactor, pyridoxal 5'-phosphate (PLP).

• Applied Microscopy
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• v.37 no.3
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• pp.167-174
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• 2007

Heat shock protein (HSP) 70 functions as a molecular chaperon and reduces stress-induced denaturation and aggregation of intracellular proteins. Erythropoietin (EPO) plays an important role during acute renal failure repair process by rapidly correcting anemia and enhancing renal tubular regeneration. The purpose of this study was to examine the effect of EPO treatment on renal HSP70 expression. Male Sprague-Dawley rats were injected rHUEPO. Kidney were preserved by in vivo perfusion with paraformaldehyde-lysine-periodate (PLP) and processed for immunohistochemistry and electron microscopy. In control kidney, HSP70 was expressed in the cortex, outer medulla and inner medulla. Especially, HSP immunoreactiviy was mainly founded in descending thin limb of outer medulla and inner medullary collecting duct. In EPO treated kidney, HSP70 expression markedly increased in the descending thin limb of outer medulla and newly detected in cortical collecting duct. Electron microscopy showed the presence of HSP immunoreactivity on the intracelluar vesicles and Golgi complex of descending thin limb and cortical collecting duct. These findings suggest that EPO treatment leads to new production of HSP70 in renal tubular cells, and induction of HSP70 by rHuEPO is causally related to protective function.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.499-510
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• 2013

Among 25 isolates, Aspergillus fumigatus ASH (JX006238) was identified as a potent producer of homocysteine ${\gamma}$-lyase. The nutritional requirements to maximize the enzyme yield were optimized under submerged (SF) and solid-state fermentation (SSF) conditions, resulting in a 5.2- and 2.3-fold increase, respectively, after the last purification step. The enzyme exhibited a single homogenous band of 50 kDa on SDS-PAGE, along with an optimum pH of 7.8 and pH stability range of 6.5 to 7.8. It also showed a pI of 5.0, as detected by pH precipitation with no glycosyl residues. The highest enzyme activity was obtained at $37-40^{\circ}C$, with a $T_m$ value of $70.1^{\circ}C$. The enzyme showed clear catalytic and thermal stability below $40^{\circ}C$, with $T_{1/2}$ values of 18.1, 9.9, 5.9, 3.3, and 1.9 h at $30^{\circ}C$, $35^{\circ}C$, $40^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$, respectively. Additionally, the enzyme $K_r$ values were 0.002, 0.054, 0.097, 0.184, and 0.341 $S^{-1}$ at $30^{\circ}C$, $35^{\circ}C$, $40^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$, respectively. The enzyme displayed a strong affinity to homocysteine, followed by methionine and cysteine when compared with non-S amino acids, confirming its potency against homocysteinuria-related diseases, and as an anti-cardiovascular agent and a specific biosensor for homocysteinuria. The enzyme showed its maximum affinity for homocysteine ($K_m$ 2.46 mM, $K_{cat}\;1.39{\times}10^{-3}\;s^{-1}$), methionine ($K_m$ 4.1 mM, $K_{cat}\;0.97{\times}10^{-3}\;s^{-1}$), and cysteine ($K_m$ 4.9 m M, $K_{cat}\;0.77{\times}10^{-3}\;s^{-1}$). The enzyme was also strongly inhibited by hydroxylamine and DDT, confirming its pyridoxal 5'-phosphate (PLP) identity, yet not inhibited by EDTA. In vivo, using Swiss Albino mice, the enzyme showed no detectable negative effects on platelet aggregation, the RBC number, aspartate aminotransferase, alanine aminotransferase, or creatinine titer when compared with negative controls.

• Journal of the Korean Society of Food Science and Nutrition
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• v.38 no.4
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• pp.470-478
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• 2009

Low trans fats were synthesized by interesterification of rice bran oil (RBO), palm stearin (PS) and high oleic sunflower seed oil (HO) using TLIM from Thermomyces lanuginosa. After 24-h reaction, physicochemical characteristics such as fatty acid and triacylglycerol composition, solid fat content, melting point, tocopherol, oryzanol and phytosterol contents were evaluated. Trans fatty acid contents of the produced low-trans fats showed less than 0.5 wt%. Mostly, triacylglycerol species in the products were palmitoyl-linoleoyl-oleoyl-glycerol (PLO), palmitoyl-oleoyl-oleoyl-glycerol (POO) and palmitoyl-oleoyl-palmitoyl-glycerol (POP). Total tocopherol contents ranged from 6.94 to 11.83 mg/100 g while $0.18{\sim}0.49$ mg/100 g of $\gamma$-oryzanol and $182.47{\sim}269.08$ mg/100 g of phytosterols were observed depending on the substrates ratios. When the content of PS in the reaction substrate was increased, solid fat content and slip melting points were increased.

• Korean Journal of Agricultural Science
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• v.39 no.3
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• pp.395-405
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• 2012

Lipozyme TL IM-catalyzed esterification was carried out to produce functional hard fat (structured lipid, SL) using palm stearin (PS) and hydrolysate of pomegranate seed oil (HPSO) of 1:6 molar ratio. HPSO contained conjugated linolenic acid (CLnA, about 80%). The reaction was performed at non-solvent system and solvent (n-hexane) system using Lipozyme TL IM (10% of total substrates, w/w) for 12, 24, and 72 hr in a shaking water bath ($55^{\circ}C$ and 185 rpm), respectively. SL synthesized in non-solvent system (NH-SL) and SL synthesized in n-hexane system (H-SL) were refined after deacidification, respectively. Their physicochemical properties were compared to obtain desirable functional hard fat. The content of CLnA in NH-SL increased from 34.38% to 40.63% with increasing reaction time. Similar results also observed in H-SL resulting in 36.81~45.83% of CLnA. In triacylglycerol (TAG) composition, the main molecules of LnLnLn (Ln=linolenic acid, PN=36) and the LnLnP (P=palmitic acid, PN=40) were newly synthesized in NH-SL and H-SL with increasing reaction time. After 72 hr reaction, iodine values of NH-SL (136.49) and H-SL (140.37) showed high values because of the high content of CLnA. Solid fat index (SFI) in NH-SL was higher than that in H-SL at each measured temperature. The predominant polymorphic forms of NH-SL and H-SL obtained after esterification for 72 hr were the desirable crystalline structure of the ${\beta}$' form.