• Journal of Periodontal and Implant Science
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• 제36권3호
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• pp.783-796
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• 2006

The periodontium is a topographically complex organ consisting of epithelial tissue, soft and mineralized tissues. Structures comprising the periodontium include the gingiva, periodontal ligament (PDL) , cementum and the alveolar bone. The molecular mechanism of differentiation in PDL fibroblast cells remain unclear. Amino acid transporters play an important role in supplying nutrition to normal and cancer cells and for cell proliferation. Amino acid transport system L is a major nutrient transport system responsible for the Na+-independent transport of neutral amino acids including several essential amino acids. The system L is divided into two major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2). In this study, the expression pattern of amino acid transport system L was, therefore, investigated in the differentiation of PDL fibroblast cells. To determine the expression level of amino acid transport system L participating in intracellular transport of amino acids in the differentiation of PDL fibroblast cells, it was examined by RT-PCR, observation of cell morphology, Alizaline red-S staining and uptake analysis after inducing experimental differentiation in PDL fibroblast cells isolated from mouse molar teeth. The results are as follows. 1. The LAT1 mRNA was expressed in the early stage of PDL fibroblast cell differentiation. This expression level was gradually reduced by differentiation- inducing time and it was not observed after the late stage. 2. The expression level of LAT2 mRNA was increased in time-dependent manner during differentiation induction of PDL fibroblast cells. 3. There was no changes in. the expression level of 4F2hc mRNA, the cofactor of LAT1 and LAT2, during differentiation of PDL fibroblast cells. 4. The expression level of ALP mRNA was gradually increased and the expression level of Col I mRNA was decreased during differentiation of PDL fibroblast cells. 5. The L-leucine transport was reduced by time from the early stage to the late stage in PDL fibroblast cell differentiation. As the results, it is considered that among neutral ammo acid transport system L in differentiation of PDL fibroblast cells, the LATl has a key role in cell proliferation in the early stage of cell differentiation and the LAT2 has an important role in the late stage of cell differentiation for providing cells with neutral amino acids including several essential amino acids.

• 식물조직배양학회지
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• 제22권3호
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• pp.137-142
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• 1995

생리적인 pH에서 음전하를 띄우고 있는 산성 아미노산인 aspartate와 glutamate는 다른 중성 아미노산과 경쟁방해 실험을 행한 연구결과에 의하면 이들도 중성 아미노산이 갖는 능동운반계와 동일한 운반자를 소유한다. 중성 아미노산을 아미노산 한 분자 당 한개의 수소이온과 동반수송되고 전하에 보상을 위해서 한 분자 당 한개의 칼륨 이온을 배출한다. 그러나 산성 아미노산은 한 분자 당 2개의 수소이온과 동반수송되고, 한 분자 당 칼륨 한 분자를 배출한다. 중성 아미노산과 같은 운반계를 소유하고, 2개의 수소이온과 동반수송되는 능동운반계는 본 실험에서 처음으로 보고된다. 이 결과로부터 두개의 동반수송된 수소이온 중 한 개는 산성 아미노산에 있는 pK$_3$카르복실기를 먼저 중화시킨 후에 중성화된 형태로 수소이온 한 분자와 동반수송된다고 사료된다. 그러므로 유채는 20개의 아미노산에 대해서 다만 2개의 운반계, 즉 일반- 과 알카리성 아미노산 운반계를 가진 것으로 확인되었다. 다른 식물에서 이미 보고된 결과들을 참고하여 아미노산 운반계의 진화적인 의미를 고찰하였다.

• 한국식품영양과학회지
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• 제34권9호
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• pp.1367-1374
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• 2005

사람의 정상 골모세포 FOB에서 아미노산 수송계 L의 발현 및 이들 수송계 L을 통한 아미노산 수송특성을 밝히기 위하여, FOB 세포에서 RT-PCR, western blot 분석 및 아미노산 uptake 실험 등을 수행하여 다음과 같은 결과들을 얻었다. FOB 세포에서 아미노산 수송계 L의 두 아형인 LAT1, LAT2및 그들의 보조인자 4F2hc의 발현을 확인할 수 있었다. FOB 세포에서 $_{L}-leucine$의 수송은 $Na^+$-비의존적이었다. FOB 세포에서 $_{L}-leucine$의 수송은 아미노산 수송계 L의 선택적 억제제인 BCH에 의해 완전히 차단되었다. FOB 세포에서 여러 아미노산들에 의한 L-leucine수송억제 실험결과는 Xenopus oocyte에서 시행되어 보고되어진 LAT1과 LAT2 수송억제 실험 결과의 특성을 모두 포함하였다. 본 연구의 결과로 사람의 정상 골모세포주인 FOB에서 는 세포성장 및 증식을 위한 중성 아미노산의 수송에 아미노산 수송계 L의 두 아형인 LAT1과 LAT2가 중요한 역할을 하고 있음을 확인할 수 있었다.

• 식물조직배양학회지
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• 제21권4호
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• pp.201-206
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• 1994

Glycine, valine, alanine과 histidine의 수송은 모든 실험된 중성 아미노산에 의해 경쟁적인 방해를 당하였다.그리고 reciprocal 연구 결과로 이들 중성 아미노산들은 서로 carrier reciprocal의 활성부위를 점유하기위해 경쟁하므로 같은 운반자를 소유한다. Histidine은 전하를 띄우지 않은 상태로 중성 아미노산 운반자를 통해서 능동수송된다. 중성 운반자의 $K_m$ 값은 아미노산이 운반자에 대한 친화성에 따라서 3가지로 분류하였다. 0.1 mM 보다 작은 값, 0.1 mM에서 0.5 mM 사이에 있는 값고 0.5 mM 보다 큰 값이다. $V_{max}\;는\;3.12mol{\cdot}h^{-1}{\cdot}g{\;}fresh{\;}weight^{-1}\;과\;15.1\;{\mu}mole{\cdot}h^{-1}{\cdot}g{\;}fresh{\;}weight^{-1}$ 사이에 있다. 중성 아미노산은 아미노산 한개당 수소이온 한개가 동반수송되고, $K^{+}$ 한개가 전하보상을 위해서 배출된다. Histidine 분자도 1 분자당 1개의 수소이온과 동반수송되나 전하를 띄운 histidine 분자로부터 수소이온 한개가 배지로 떨어져 나오므로 동반수송된 수소이온의 움직임을 일시적으로 측정 할 수 없다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제32권3호
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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.

• 대한약리학회:학술대회논문집
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• 대한약리학회 2006년도 The 6th Congress of the Federation of Asian and Oceanian Physiological Societies
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• pp.91.2-91.2
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• 2006

• Archives of Pharmacal Research
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• 제28권4호
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• pp.421-432
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• 2005

In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the functional characterizations and the localization in human kidney. Northern blot analysis showed that hLAT2 mRNA was expressed at high levels in the heart, brain, placenta, kidney, spleen, prostate, testis, ovary, lymph node and the fetal liver. The hTAT1 mRNA was detected at high levels in the heart, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus and prostate. Immunohistochemical analysis on the human kidney revealed that the hLAT2 and hTAT1 proteins coexist in the basolateral membrane of the renal proximal tubules. The hLAT2 transports all neutral amino acids and hTAT1 transports aromatic amino acids. The basolateral location of the hLAT2 and hTAT1 proteins in the renal proximal tubule as well as the amino acid transport activity of hLAT2 and hTAT1 suggests that these transporters contribute to the renal reabsorption of neutral and aromatic amino acids in the basolateral domain of epithelial proximal tubule cells, respectively. Therefore, LAT2 and TAT1 play essential roles in the reabsorption of neutral amino acids from the epithelial cells to the blood stream in the kidney. Because LAT2 and TAT1 are essential to the efficient absorption of neutral amino acids from the kidney, their defects might be involved in the pathogenesis of disorders caused by a disruption in amino acid absorption such as blue diaper syndrome.

• Journal of Microbiology
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• 제33권2호
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• pp.142-145
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• 1995

The pumb gene encoding a basic amino acid transport protein in Neurospora crassa could be cloned by using a mutant strain defective in pmb gene as a host strain, using a negative selection on the media containing amino acid analogue canavanine. To select positive transformants of the genes for cloning, an auxotrophic marker (his-2) was added to a pmb mutant strain by mating ; a triple mutant (pmn : pmb : his-2) was constructued by crossing a strain defective in basic amino acid transport system (# 1683-bat um 535 "A") to a double mutant strain defective in neutral amino acid transport and histidine production (mitrol : his-2 "a"). Crossing was performed on synthetic crossing (SC) media containing histidine. The pmn : pmb and pmn :pmb : his-2 strains were selected among the progeny colonies from crosses on plates containing 5- .mu.g/ml para-fluoro-phenylalanine (PFPA), 200 .mu.g/ml canavanine, and 500 .mu.g/ml histidine. The selected colonies were cultured on minimal media with or without histidine for discarding pmn : pmb strain, because the pmn : pmb : his -2 strain grows only on histidine containing media. The pmn :pmb : his-2 strain selected can be used as a host strain for the cloning of the pmb and the pmn genes from a Neurospora genomic library by means of positive selections.

• The Korean Journal of Physiology and Pharmacology
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• 제1권2호
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• pp.185-193
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• 1997

The characteristics of $Na^+$-dependent cycloleucine uptake was investigated in OK cells with regard to substrate specificity and regulation by protein kinase C (PKC). Inhibition studies with different synthetic and natural amino acids showed a broad spectrum affinity to neutral amino acids regardless of their different side chains including branched or aromatic, indicating that the $Na^+$-dependent cycloleucine uptake in OK cells is mediated by System $B^o$ or System $B^o$-like transporter rather than the classical System A or ASC. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate, but not $4{\alpha}-PMA$ elicited a time-dependent biphasic stimulation of $Na^+$-dependent cycloleucine uptake, which produced early transient peak at 30 min and late sustained peak at 180min. Both the early and late stimulations by PMA were due to an increase in Vmax and not due to a change in Km. PKC inhibitors blocked both the early and late stimulation by PMA, while protein synthesis inhibitors blocked the late stimulation only. These results suggest the existence and regulation by PKC of System $B^o$ or System $B^o$-like broad spectrum transport system for neutral amino acids in OK cells.

• 한국식품영양과학회지
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• 제37권5호
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• pp.555-560
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• 2008

사람 두경부 편평세포암종 HEp2 세포를 이용하여 아미노산 수송계 L 억제제인 BCH의 암세포 성장억제에 미치는 효과와 세포성장 억제기전을 밝히기 위해 HEp2 세포에서 uptake 실험, MTT 분석, DNA fragmentation 분석 및 immunoblotting 등을 시행하여 다음과 같은 결과를 얻었다. 아미노산 수송계 L 억제제인 BCH는 L-leucine uptake를 농도 의존적으로 억제하였으며, 그 $IC_{50}$은$ 51.2{\pm}3.8{\mu}M$로 산출되었다. BCH는 HEp2 세포의 성장을 시간과 농도에 의존적으로 억제하였다. BCH를 처리한 실험군에서 DNA fragmentation 현상은 볼 수 없었다. BCH를 처리한 실험군에서 procaspase-3과 procaspase-7의 proteolytic cleavage 현상은 볼 수 없었다. 본 연구의 결과로서 사람 두경부 편평세포 암종 HEp2 세포에서 아미노산 수송계 L 억제제 BCH는 LAT1 활성을 억제하여 세포성장에 필수적인 L-leucine 등 중성아미노산의 세포 내 고갈을 유도함으로써 HEp2 세포의 성장억제를 유도할 가능성이 있는 것으로 사료된다.