• Title/Summary/Keyword: Amino acid transporter

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Ionomycin Recovers Taurine Transporter Activity in Cyclosporin A Treated macrophages

  • Kim, Ha-Won;Lee, Eun-Jin;Kim, Won-Bae;Hyun, Jin -Won;Kim, Byung-Kak
    • Preventive Nutrition and Food Science
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    • v.4 no.4
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    • pp.265-269
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    • 1999
  • Taurine is a major $\beta$-amino acid in various tissues. Taurine transporter (TAUT) is responsible for the transportation of taurine in the cell. The transporter is affected by various stimuli to maintain its cell volume. Macrophage cell volume varies in its activated states. In our experiment, it was found that the murine macrophage cell line, RAW264.7, expressed TAUT protein in its membrane. Its transportation activities could be blocked by a $\beta$-amino acid such as $\beta$-alanine, but not by $\alpha$-amino acids in this cell line. When assessed in RAW264.7 under the influence of immunosuppressive reagents, the activity of the TAUT was decreased by the treatment of rapamycin (RM) or cyclosporin A (CsA). However when ionomycin (IM) was added to this system, TAUT activity was recovered only in CsA-treated cells in a concentration-dependent manner. In order to inhibit the voltage gated {TEX}$Ca^{+2}${/TEX} channel, calmidazolium was added to the RAW264.7 cell line. Treatment of the cell with calmidazolium completely blocked TAUT. Furthermore, addition of IM to this system recovered the activity of TAUT again. When we added phorbol myristate acetate (PMA) to the cell line, secretion of nitric oxide (NO) was increased 4-fold and the TAUT activity was decreased 5-fold. However, the addition of N-nitro L-arginine methyl ester (L-NAME), an inducible NO synthase (iNOS) inhibitor, to the PMA-treated cells, resulted in the recovery of TAUT activity. These results showed that the activity of TAUT was sensitive to the intracellular concentrations of both {TEX}$Ca^{+2}${/TEX} and NO.

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EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF AMINO ACID TRANSPORT SYSTEM L IN SAOS2 HUMAN OSTEOGENIC SARCOMA CELLS (사람 골육종 세포 Saos2에서 아미노산 수송계 L의 발현 및 기능적 특성)

  • Kim, Su-Gwan;Kim, Hyun-Ho;Kim, Chang-Hyun;Kim, Do-Kyung
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.32 no.3
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    • pp.200-208
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    • 2006
  • Amino acids are required for protein synthesis and energy sources in all living cells. The amino acid transport system L is a major nutrient transport system that is responsible for $Na^+$-independent transport of neutral amino acids including several essential amino acids. In malignant tumors, the L-type amino acid transporter 1 (LAT1), the first isoform of system L, is highly expressed to support tumor cell growth. In the present study, the expression and functional characterization of amino acid transport system L were, therefore, investigated in Saos2 human osteogenic sarcoma cells. RT-PCR and western blot analyses have revealed that the Saos2 cells expressed the LAT1 and the L-type amino acid transporter 2 (LAT2), the second isoform of system L, together with their associating protein heavy chain of 4F2 antigen (4F2hc) in the plasma membrane, but the expression of LAT2 was very weak. The uptakes of [${14}^C$]L-leucine by Saos2 cells were $Na^+$-independent and were completely inhibited by the system L selective inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The affinity of [${14}^C$]L-leucine uptake and the inhibition profiles of [${14}^C$]L-leucine uptake by various amino acids in the Saos2 cells were comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [${14}^C$]L-leucine uptake is, therefore, mediated by LAT1 in the Saos2 cells. These results suggest that the transports of neutral amino acids including several essential amino acids into Saos2 human osteogenic sarcoma cells are for the most part mediated by LAT1. Therefore, the Saos2 human osteogenic sarcoma cells are excellent tools for examine the properties of LAT1. Moreover, the specific inhibition of LAT1 in tumor cells might be a new rationale for anti-tumor therapy.

Effect of hyperthermia on cell viability, amino acid transfer, and milk protein synthesis in bovine mammary epithelial cells

  • Zhou, Jia;Yue, Shuangming;Xue, Benchu;Wang, Zhisheng;Wang, Lizhi;Peng, Quanhui;Hu, Rui;Xue, Bai
    • Journal of Animal Science and Technology
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    • v.64 no.1
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    • pp.110-122
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    • 2022
  • The reduction of milk yield caused by heat stress in summer is the main condition restricting the economic benefits of dairy farms. To examine the impact of hyperthermia on bovine mammary epithelial (MAC-T) cells, we incubated the MAC-T cells at thermal-neutral (37℃, CON group) and hyperthermic (42℃, HS group) temperatures for 6 h. Subsequently, the cell viability and apoptotic rate of MAC-T cells, apoptosis-related genes expression, casein and amino acid transporter genes, and the expression of the apoptosis-related proteins were examined. Compared with the CON group, hyperthermia significantly decreased the cell viability (p < 0.05) and elevated the apoptotic rate (p < 0.05) of MAC-T cells. Moreover, the expression of heat shock protein (HSP)70, HSP90B1, Bcl-2-associated X protein (BAX), Caspase-9, and Caspase-3 genes was upregulated (p < 0.05). The expression of HSP70 and BAX (pro-apoptotic) proteins was upregulated (p < 0.05) while that of B-cell lymphoma (BCL)2 (antiapoptotic) protein was downregulated (p < 0.05) by hyperthermia. Decreased mRNA expression of mechanistic target of rapamycin (mTOR) signaling pathway-related genes, amino acid transporter genes (SLC7A5, SLC38A3, SLC38A2, and SLC38A9), and casein genes (CSNS1, CSN2, and CSN3) was found in the heat stress (HS) group (p < 0.05) in contrast with the CON group. These findings illustrated that hyperthermia promoted cell apoptosis and reduced the transport of amino acids into cells, which inhibited the milk proteins synthesis in MAC-T cells.

Pharmacogenomic Application for Gene Targeting and Molecular Characterization of a Nucleoside Transporter hCNT2 in Human Intestine

  • Shin, Ho-Chul;Lee, Jong-Hwa;Lee, Han-Ok;Duxin Sun;Gordon L. Amidon
    • Proceedings of the Korean Society of Toxicology Conference
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    • 2003.10b
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    • pp.158-159
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    • 2003
  • We have cloned and functionally expressed a sodium dependent human nucleoside transporter, hCNT2, from a CNS cancer cell line U251. Our cDNA clone of hCNT2 had the same predicted amino acid sequence as the previously cloned hCNT2 transporter. Of the several cell lines studied, the best hCNT2 transport function was obtained when transiently expressed in U251 cells.(omitted)

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Effects of coated cysteamine hydrochloride on muscle fiber characteristics and amino acid composition of finishing pigs

  • Bai, Miaomiao;Liu, Hongnan;Xu, Kang;Yu, Rong;Oso, Abimbola Oladele;Deng, Jinping;Yin, Yulong
    • Asian-Australasian Journal of Animal Sciences
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    • v.32 no.9
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    • pp.1430-1438
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    • 2019
  • Objective: This experiment was designed to determine the effects of coated cysteamine hydrochloride (CC) on muscle fiber characteristics, amino acid composition and transporters gene expression in the longissimus dorsi muscle (LDM) of finishing pigs. Methods: Two hundred and sixteen Duroc/Landrace/Yorkshire cross-bred male finishing pigs were fed with a corn-soybean basal diet supplemented with 0, 70, and 140 mg/kg cysteamine. Each group contained eight replicates of nine pigs per replicate. After 29 days, one pig was randomly selected from each replicate and slaughtered. Blood and LDM samples were collected and analyzed. Results: The results showed that supplemental dietary CC increased (p<0.05) the muscle fiber density. And CC supplementation also up-regulated (p<0.05) the expression of myosin heavy chain 1 (MyHC1) and MyHC2x mRNA levels, and down-regulated (p<0.05) MyHC2b expression in the LDM. Additionally, supplemental dietary CC reduced (p<0.05) the concentration of total cholesterol in the plasma and enhanced (p<0.05) the concentrations of essential amino acid and total amino acid in the LDM. The relative expression levels of chloramphenicol acetyltransferase 2, $b^{0,+}$ amino acid transporter, and $y^+$-L-type amino acid transporter 1 were upregulated (p<0.05) in the LDM when pigs were fed with the dietary CC of 70 mg/kg. Conclusion: Cysteamine supplementation could increase fiber density and distribution of fiber types. It also improved the deposition of protein in the LDM by up-regulated the expression of amino acid transporters.

Molecular Mechanism of ABC Transporter Mdr49A Associated with a Positive Cross-Resistance in Transgenic Drosophila (형질전환 초파리를 이용한 Mdr49A 유전자의 살충제 교차저항성 기능 구명)

  • Seong, Keon Mook;Pittendrigh, Barry R.
    • Korean journal of applied entomology
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    • v.59 no.4
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    • pp.341-348
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    • 2020
  • The ATP-binding cassette (ABC) transporter superfamily represents the largest transmembrane protein that transports a variety of substrates across extra- and intra-cellular membranes. In insects, the ABC transporter proteins play crucial roles in insecticide resistance. To date, no studies have investigated the involvement of ABC transporter gene for cross-resistance to insecticide chemistries. Here, we studied such possible mechanisms against six conventional insecticides using transgenic Drosophila melanogaster strains carrying Mdr49 transcript variant A. For the 91-R and 91-C strains of Drosophila melanogaster, although they have a common origin, 91-R has been intensely selected with DDT for over 60 years, while 91-C has received no insecticide selection. Our transgenic analyses showed that overexpression of 91-R-MDR49 transcript variant A along with three amino acid variations can yield a relatively low degree of cross-resistance to carbofuran (2.0~6.7-fold) and permethrin (2.5~10.5-fold) but did not show cross-resistance to abamectin, imidacloprid, methoxychlor, and prothiofos as compared to the Gal4-driver control strain without transgene expression. These results indicate that the overexpression of Mdr49A in itself leads to a cross-resistance and three amino acid changes have additional effects on positive cross-resistance to carbofuran and permethrin.

Isolation and characterization of LHT-type plant amino acid transporter gene from Panax ginseng Meyer

  • Zhang, Ru;Zhu, Jie;Cao, Hong-Zhe;Xie, Xiao-Lei;Huang, Jing-Jia;Chen, Xiang-Hui;Luo, Zhi-Yong
    • Journal of Ginseng Research
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    • v.37 no.3
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    • pp.361-370
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    • 2013
  • A lysine histidine transporter (LHT) cDNA was isolated and characterized from the roots of Panax ginseng, designated PgLHT. The cDNA is 1,865 bp with an open reading frame that codes for a protein with 449 amino acids and a calculated molecular mass of 50.6 kDa with a predicted isoelectric point of 8.87. Hydropathy analysis shows that PgLHT is an integral membrane protein with 9 putative membrane-spanning domains. Multiple sequence alignments show that PgLHT shares a high homology with other plant LHTs. The expression profile of the gene was investigated by real-time quantitative polymerase chain reaction during various chemical treatments. PgLHT was up-regulated in the presence of abscisic acid, salicylic acid, methyl jasmonate, NaCl, and amino acids. To further explore the function of PgLHT gene, full-length cDNA of PgLHT was introduced into P. ginseng by Agrobacterium rhizogenes A4. The overexpression of PgLHT in the hairy roots led to an obviously increase of biomass compared to the controls, and after addition of the amino acids, the overexpressed-PgLHT hairy roots grew more rapidly than untreated controls during early stage of the culture cycle. The results suggested that the PgLHT isolated from ginseng might have role in the environmental stresses and growth response.

Gene Expression of Taurine Transporter and Taurine Biosynthetic Enzyme During Embryonic Development

  • Yoon, Seyng-Hyun;Kim, Ha-Won
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 2003.11a
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    • pp.87-87
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    • 2003
  • Taurine (2-aminoethanesulfonic acid, $^{+}NH_3CH_2CH_2{SO_3}^{-}$) is endogenous $\beta$-amino acid which is essential in fetal nutrition and development and is present in abundant quantities in several tissues of fetus. In utero, taurine deficiency causes abnormal development and abnormal function of brain, retina, kidney and myocardium. Thus, transfer of taurine into fetus is important during embryonic development. Taurine transporter (TauT) has 12 hydrophobic membrane -spanning domains, which is typical of the $Na^{+}$- and $Cl^{-}$-dependent transporter gene family. Among the various biosynthetic enzymes of taurine, cysteine sulfinic acid decarboxylase (CSD) is the rate-limiting enzyme for biosynthesis of taurine. However, the enzyme activities of taurine biosynthesis are limited in early stage of embryonic development. To analyze the expression period of TauT and CSD during embryonic development, we have investigated the gene expression of TauT and CSD using reverse transcriptase polymerase chain reaction (RT-PCR) in mouse and chicken embryos. RT-PCR anaylsis revealed that both TauT and CSD mRNAs were already expressed at Day-4.5 in mouse embryo. In chicken whole embryo, TauT and CSD mRNAs began to appear on developing times of 48 hrs and 12 hrs, respectively. TauT mRNA was detected in the organs of heart, brain and eye of the day-3 chicken embryo. Our data show that TauT and CSD mRNAs were expressed in early stage of embryonic development.

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