• 한국식품영양과학회지
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• 제32권5호
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• pp.758-763
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• 2003

구강 편평 세포암종 KB 세포를 이용하여 아미노산 수송계 L억제제인 BCH의 세포 성장억제에 미치는 효과를 밝히기 위해, KB세포에서 uptake실험, MTT분석 및 DNA frag-mentation분석 등을 시행하여 다음과 같은 결과를 얻었다. KB 세포에서는 아미노산 수송계 L 중에서 LAT1과 그 보조인자 4F2hc를 통해 BCH및 중성 아미노산들이 수송되었다. BCH는 시간과 농도에 의존적으로 KB세포의 성장을 억제시켰다. BCH를 처리한 실험군에서 DNA fragmentation 현상은 볼 수 없었다. 본 연구의 결과로서 구강암 세포주인 구강 편평세포암종 KB 세포에서 LAT1과 그 보조인자 4F2hc를 통해 BCH및 중성 아미노산의 수송이 이루어지고 있다는 것을 확인할 수 있었으며 BCH는 이 LAT1을 차단하여 중성 아미노산들의 세포 내 고갈을 유도함으로서 KB 세포 성장의 억제를 유도하는 것으로 사료된다.

• Journal of Pharmaceutical Investigation
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• 제20권4호
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• pp.223-228
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• 1990

The contribution of endogenous transport systems to the blood-brain barrier (BBB) transport of basic and acidic drugs was studied by using a carotid injection technique in rats and an isolated bovine cerebrovascular disease state were compared between the normotensive rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) which have been well established as an animal model with pathogenic similarities to humans. Basic drugs such as eperisone, thiamine and scopolamine inhibited, in a concentration dependent manner the in vivo uptake of $[{^3}H]choline$ through BBB, whereas amino acids and acidic drugs such as salicylic acid and valproic acid did not inhibit the uptake. The uptake of $[^3H]choline$ by B-CAP increased with time and showed a remarkable temperature dependency. The uptake of $[^3H]choline$ by B-CAP showed the very similar inhibitory effects as observed in the in vivo brain uptake, and was competitively inhibited by a basic drug, eperisone. The in vivo BBB uptakes of $[^3H]acetic$ acid and $[^{14}C]salicylic$ acid were dependent on pH of the injectate and the concentration of drugs. Several acidic drugs such such as salicylic acid, benzoic acid and valproic acid inhibited the in vivo uptake of $[^3H]acetic$ acid, whereas amino acid, choline and a basic drug such as eperisone did not inhibit the uptake. The uptake of acetic acid by B-CAP was competitively inhibited by salicylic acid. The permeability surface area product (PS) through BBB for $[^3H]choline$ in SHRSP was significantly lower than that in WKY. The concentration of choline in the brain dialysate in SHRSP was about half of that in WKY, while no significant difference was observed in the plasma concentration of choline between SHRSP and WKY. No significant difference was observed in the transport of monocarboxylic acids, glucose and neutral amino acid through BBB between SHRSP and WKY. From these results, it was concluded that BBB transport system of choline contributes to the transport of basic drugs through BBB, that acidic drugs can be transported via a moncarboxylic acid BBB transport system and that the specific dysfuntion of the BBB choline transport in SHRSP was ascribed to the reduction of the maximum velocity of choline concentration in the brain interstitial fluids.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.154-161
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• 2024

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a nonessential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [¹⁴C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1^G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [¹⁴C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [¹⁴C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [¹⁴C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

• Journal of Animal Science and Technology
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• 제64권1호
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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.

• 한국식품영양과학회지
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• 제33권9호
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• pp.1451-1456
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• 2004

사람의 여러 암세포주들에서 아미노산 수송체 LAT1 및 그 보조인자 4F2hc mRNA의 발현과 LAT1의 대표적 기질인 L-leucine 수송을 조사하여 이들 사이의 상관관계를 밝히기 위해, 암세포주들에서 northern blot analysis 및 uptake 실험 등을 시행하여 다음과 같은 결과를 얻었다. 조사한 26 종류의 사람 암 유래 세포주들에서 LAT1 mRNA의 뚜렷한 발현을 확인할 수 있었으나 그 발현양에는 차이가 있었다. 조사한 26종류의 사람 암 유래 세포주들에서 4F2hc mRNA의 뚜렷한 발현을 확인할 수 있었으며, LAT1의 경우에서와 같이 발현하는 정도가 조금씩 다름을 확인할 수 있었다. 사람의 암 유래 세포주들에서 아미노산 transporter에 의한 L-[$^{14}C$]leucine 수송을 확인할 수 있었다. 사람의 암세포주들에서 LAT1 mRNA의 발현정도가 높을수록 L-[$^{14}C$]leucine의 수송능력이 증가함을 알 수 있었다. 사람의 암세포주들에서 4F2hc mRNA의 발현정도가 높을수록 L-[$^{14}C$]leucine 수송 능력이 증가하는 경향은 보였으나, 통계적인 유의성은 없었다. 본 연구의 결과에 의해 사람 암세포주들에서 아미노산 transporter LAT1의 발현과 아미노산 L-leucine의 수송 사이와의 상관관계를 확인할 수 있었으며, 아미노산 transporter LAT1의 특이적 억제에 의한 암세포의 성장 억제에 관한 또 하나의 방향성을 제시할 수 있을 것으로 사료된다.

• Journal of Nutrition and Health
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• 제18권3호
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• pp.209-216
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• 1985

임신말 여성의 태반을 중심으로 모체와 태아사이의 아미노산 이동 현상을 이해하고저 모체쪽의 antecubital vein, uterine vein과 iliac artery에서 태아쪽의 umbilical vein과 artery에서 혈액을 채취하여 23개의 혈장 유리아미노산 농도를 측정 비교하였다. 본 실험에서 얻어진 결과들을 요약하면 다음과 같다. 첫째, glutamate를 제외하고는 모체의 antecubital vein과 태아의 umbilical vein의 아미노산 농도의 비는 1.21에서 3.21의 범위를 보여주고 있어 태아의 혈장 유리아미노산 농도가 모체에서 보다 훨씬 높았다. 둘째, 모체쪽의 iliac artery의 아미노산 농도와 태아의 umbilical vein의 아미노산 농도 사이에 존재하는 상호관계를 살펴보았을 때, 대부분의 아미노산들은 직선의 관계를 보였다. 이와같은 결과는 직선의 기울기가 1에 가까운 중성 아미노산은 단순확산, 직선의 기울기가 1과 상이한 염기성 및 산성아미노산은 단순확산과 능동적 이동과의 동적평형으로 해석되었으며, 따라서 태반은 아미노산에 대하여 단순한 장벽만의 역할을 하는 것이 아님을 의미하고 있다.

• 대한화학회지
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• 제37권1호
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• pp.10-21
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• 1993

2-hydroxyethyl methacrylate(HEMA)을 중합한 P(HEMA)막을 통하여 10가지 아미노산들의 확산계수, 분배계수 및 투과도를 용액의 pH 변화하에서 조사 연구하였다. 아미노산의 투과도는 각 아미노산들의 분자형태, 분자크기 및 전하량에 따라 다른 값을 나타냄을 확인하였으며, 특히 용액의 pH에 따라 전하량이 변화하는 각 아미노산은 P(HEMA)막을 통하여 투과시킬 때 모든 아미노산이 중성영역용액에서 가장 많이 투과되고 이때 막은 미세공막으로 작용하며, 염기성영역용액에는 산성영역용액보다 더 많이 투과되고 막은 분배막으로 작용함을 알았다.

• 한국미생물·생명공학회지
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• 제51권1호
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• pp.37-42
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• 2023

Neutral and non-essential amino acid, glutamine (Gln), plays an essential role in supplying nitrogen to all the amino acids and nucleotides in the mammalian body. Gln is also the most important carbon source that provides intermediates for gluconeogenesis and fatty acid synthesis and supplements the tricarboxylic acid cycle in fast-growing cancer cells. Among the known 14 Gln transporter genes, soluted carrier family 1 member 5 (SLC1A5) has been reported to be closely associated with cancer cell growth. Three variants (v1, v2, and v3) have been derived from SLC1A5. Here, we established a heterologous gene expression system for the active form of human SLC1A5 variant-2 (hSLC1A5v2) in Escherichia coli. v2 is the smallest variant that has not yet been studied. Four expression systems were investigated: pBAD, pCold, pET, and pQE. We also addressed the problem of codon usage bias. Although pCold and pET overexpressed hSLC1A5v2 in E. coli, they were functionally inactive. hSLC1A5v2 using the pBAD system was able to catalyze the successful transport of Gln, even if it was not highly expressed. Initial activity of hSLC1A5v2 for [¹⁴C] Gln uptake in E. coli reached up to 6.73 μmole·min^-1·gDW^-1 when the cell was induced with 80 mM L-arabinose. In this study, we demonstrated a heterologous expression system for the human membrane protein, SLC1A5, in E. coli. Our results can be used for the functional comparison of SLC1A5 variants (v1, v2, and v3) in future studies, to facilitae the developement of SLC1A5 inhibitors as effective anticancer drugs.