Characterization of PCR fragment of metallothionein gene from liver mRNS of channel catfish

송영환,Song, Young-Hwan 한국어병학회 1997 한국어병학회지 Vol.10 No.1

Metallothionein은 세균에서 척추동물에 이르기 까지 모든 생명체에 존재하며, 중금속의 세포내농도를 조절하는 중요한 단백질이다. 현재까지 metallothionein의 기능 및 유도기작에 관한 연구는 많이 진척되지는 않았으나, 여러 metallothionein 유전자의 구조가 밝혀져 있는 실정이다. 특히 어류의 metallothionein은 여러종류의 중금속과 환경적인 자극에 의하여 유도되고 정량적인 RT-PCR의 방법으로 metallothionein 유전자의 RNA transcript를 측정함으로써 환경적인 자극의 정도와 중금 속의 상대적인 양을 측정할 수 있기 때문에 중요한 단백질로 인식되고 있다. 본 연구에서는 유전자내부의 특이적 primer와 통상적인 3`말단의 primer를 이용하여 PCR에 의해 450 bp에 해당하는 metallothionein 유전자의 일부의 특성을 조사하였다. 챠넬메기의 cDNA library로부터 PCR에 의해 증폭된 450 bp의 PCR 절편은 다른 어류의 metallothionein 유전자와는 유사성을 보이지 않았다. Metallothionein is an important and essential protein to control the intracellular concentration of heavy metals, which exist in all organisms from bacteria to vertebrates. Although the detailed functions and induction mechanisms of metallothionein gene have not been clearly characterized until yet, the structure of several metallothionein genes has been revealed. Especially, piscine metallothionein is regarded as an important protein because it is induced by several heavy metal pollutants and environmental stress and it could be determined the comparative amount of heavy metals and the extent of environmental stress by assaying the RNA transcript of metallothionein gene in the method of the quantitative RT-PCR(Reverse Transcriptase Polymerase Chain Reaction). In this study I have characterized the 450 bp PCR fragment of metallothionein gene amplified by using the mixture of internal specific primers and universal 3' end primer. The nucleotide sequence analysis of 450 bp PCR fragment amplified in cDNA library of channel catfish did not show strong homology to other piscine metallothionein genes.

Effect of Chlorella on Metallothionein Synthesis and Binding Capacity of Cadmium in Cadmium Poisoned Rat Liver and Kidney

Hwang Yoo-Kyeong,Choi Hyun-Jin,Nan Meng,Yoo Jai-Du,Kim Yong-Ho 대한의생명과학회 2006 Biomedical Science Letters Vol.12 No.1

The rate of metallothionein synthesis on cadmium-poisoned rats reflects the level of toxicity, and also it reduces the toxicity which is caused by the uptake of cadmium. Chlorella supplementation in the diets of the cadmium-poisoned rats decreased the concentration of cadmium in blood and urine compared with the control group. Although the liver and kidneys of rats are major target organs of cadmium and coherence of metallothionein and cadmium, no previous study has determined the correlation between the rate of metallothionein synthesis in the liver and kidneys of rats and dietary supplementation of chlorella with cadmium uptake. This study analyzed total metallothionein level on the tissue of the liver and kidneys, the concentration of cadmium bound to the metallothionein, and the total concentration of cadmium on the tissue of the liver and kidneys after dietary supplementation with 1%, 5%, and 10% dried chlorella and 40 ppm of cadmium to 46 male SD rats (mean weight: $150\pm20\;g$) for 4 weeks. According to the data analysis of the total rate of metallothionein synthesis in the liver and kidneys, the group of SD rats on the supplementation with 1% chlorella and 40 ppm of cadmium showed a rate of $93.2\pm8.9\;ng/g$, a significant decrease of 58.8% compared to that of the control group of SD rats on the supplementation with cadmium only, which showed a rate of $227.3\pm32.5 ng/g$ (P=0.0001). In contrast, no significant difference was observed through the changing of chlorella concentrations between 5% and 10% chlorella supplementation with cadmium. The group supplemented with 1% or greater chlorella levels represented a greater decrease in the total cadmium concentration of the kidney and liver tissues, the amount of total metallothionein synthesis, the amount of metallothionein with binding to cadmium, and the concentration of free cadmium without binding to metallothionein. Consequently, the supplementation of 1% and 5% chlorella was effective in reducing the synthesis of metallothionein for cadmium uptake, but increased the rate of binding of cadmium to metallothionein.

챠넬메기의 metallothionein cDNA 유전자의 cloning 및 그 특성에 관한 연구

이인정,송영환,Lee, In-Jung,Song, Young-Hwan 한국어병학회 1992 한국어병학회지 Vol.5 No.2

Metallothionein은 세포내의 중금속의 농도을 조절하는 주요한 단백질로서 bacteria에서 척추동물에 이르기까지 모든 생명체에서 나타나는 공통된 단백질이다. 비록 metallothionein의 정확한 기능은 알려져 있지 않으나 독성을 나타내는 중금속에 대하여 세포내 방어기작에 관여할 뿐만 아니라 여러다른 유전자의 총괄적 조절기작 및 matalloprotein의 발현에 관여할 것으로 보고있다. 본 연구에서는 Channel Catfish의 metallothionein cDNA 유전자를 poly(A)를 갖는 mRNA로 부터 Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR)에 의하여 cloning하였다. 증폭된 PCR products는 pBluescript SK+의 EcoRV site 및 pUC19의 Smal site에 dT tailing을 하여 cloning하였으며, PCR products는 multicloning site에 있는 EcoRI 및 HindIII 로 절단하여 확인하거나 신속한 PCR screening에 의하여 확인하였다. 여러 PCR clone 중 하나인 pMT150에 대한 DNA 염기서열을 조사한 결과 다른 어류의 metallothionein cDNA 유전자와 높은 유사성을 보였다. Metallothionein is an essential and common protein to regulate the intracellular concentration of heavy metals, which exist in most organisms from bacteria to vertebrates. Although the detailed function of metallothianein has not been fully identified until yet, it may be involoved in the cellular protection against the heavy metal toxicity and in the global regulation of several other genes and the expression of metalloproteins. We have cloned the full cDNA clone of metallothionein gene in Channel Catfish by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) starting from poly(A)-containing mRNAs. All PCR fragments have been subcloned into EcoRV site of pBluescript SK+ and dT-tailed at Smal site of pUC19, then PCR products are recovered by the double digestion of recombinant plasmids wiht EcoRI and HindIII, which are adjacent to EcoRV site in multicloning sites or by rapid PCR screening. The nucleotide sequence analysis of pMT150(one of the PCR clones) showed high homology with several other piscine metallothionein cDNA genes.

미호종개 metallothionein 유전자의 구조 및 중금속 노출과 고온 자극에 대한 MT mRNA의 발현 특징 분석

이상윤 ( Sang Yoon Lee ),남윤권 ( Yoon Kwon Nam ) 한국어류학회 2011 韓國魚類學會誌 Vol.23 No.1

멸종위기 어류 미호종개(Cobitis choii)로부터 중금속해독 단백질(metallothionein) 유전자를 분리, 클로닝하고 중금속 및 고온 스트레스에 대한 전사 발현 특징을 분석하였다. 미호종개 metallothionein는 gDNA, mRNA 및 아미노산 서열 모두에서 경골 어류 MT들의 구조적 특징을 잘 보전하고 있었으며, 생물정보분석을 통해 미호종개 MT 유전자 5`-upstream 영역은 중금속 조절, 면역 반응 및 온도 반응에 관여하는 다양한 전사 조절인자들의 부착 위치들을 포함하는 것으로 관찰되었다. 카드뮴(Cd), 구리(Cu), 니켈(Ni), 망간(Mn) 및 아연(Zn)을 이용한 침지 노출 실험(0.5 및 1.0 μM; 24시간)에서 미호종개 MT mRNA 발현은 구리 및 카드뮴 처리군에서 가장 많이 유도되었고(1.0 μM Cu 처리군에서 최대 10배), 망간 처리군에서는 비교적 적은 양의 MT 발현이 유도된 반면(2배), 아연 및 니켈 노출 군에서는 유의적인 MT 발현의 증감이 관찰되지 않았다. 또한 미호종개 MT 전사 발현은 고온 자극(25°C로부터 31°C까지 증가)에도 민감하게 반응하는 것으로 나타나, 31°C 도달시점에서 25°C 초기 MT mRNA 발현 수준보다 9배 높은 mRNA 발현이 관찰되었다. 본 연구 결과는 MT 기반의 유전자 발현 분석을 이용함으로써, 향후 멸종위기 어류 미호종개의 스트레스 반응 연구에 유용한 기초 자료를 제공할 수 있다고 기대된다. Gene and promoter structures of metallothionein (MT) from Miho spine loach (Cobitis choii; Cypriniformes) were characterized, and the transcriptional responses to experimental exposures to heavy metals and heat stress were examined. The C. choii metallothionein displayed well-conserved features of teleostean metallothioneins at gDNA, mRNA and amino acid levels. Bioinformatic analysis predicted that the C. choii MT regulatory region potentially possessed various motifs or elements targeted by various transcription factors associated with metal-coordinating regulation (e.g., metal transcription factor-1), immune responses (e.g., nuclear factor kappa B), and thermal modulations (e.g., heat shock factor). Acute heavy-metal exposures to 0.5 or 1.0 μM of cadmium (Cd), copper (Cu), manganese (Mn), nickel (Ni) or zinc (Zn) showed that MT transcription was significantly stimulated by Cd (9.6-fold relative to non-exposed control) and Cu (10.4-fold), only moderately by Mn (2.4-fold), but hardly by Ni and Zn. Elevation of water temperature from 25°C to 31°C caused a rapid modulation of MT mRNAs toward upregulation to 9.5-fold; however, afterward the elevated mRNA level slightly decreased during further incubation at 31°C for 6 h. Results from this study suggest that MT-based expression assay could be a useful basis for better understanding the metal- and/or heat-caused stresses in this endangered fish species.

Metallothionein이 지질과산화반응과 Aldehyde Oxidase활성에 미치는 영향

허근,신억섭,박종민,Huh, Keun,Shin, Uk-Seob,Park, Jong-Min 한국독성학회 1995 Toxicological Research Vol.11 No.1

The effects of metallothionein and cadmium ion on the hepatic aldehyde oxidase activity and brain lipid peroxidation were tested in vitro. The content of brain lipid peroxide at the condition of normal or $300{\mu}M$ Fe(II)-induced was remarkably reduced by the addition of metallothionein in the incubation mixture. The induced content of lipid peroxide by cadmium $(30{\mu}g/ml)$ ion was reduced by metallothionein $(100{\mu}g/ml)$. The activity of aldehyde oxidase was not affected by metallothionein, but cadmium ion $(8.38{\mu}g/ml)$ increased the activity of aldehyde oxidase about 80% compared to the control. The cadmium-induced activity of aldehyde oxidase was restored to the control level by metallothionein or penicillamine.

Metallothionein이 지질과산화반응과 Aldehyde Oxidase활성에 미치는 영향

허근,신억섭,박종민 영남대학교 약품개발연구소 1996 영남대학교 약품개발연구소 연구업적집 Vol.6 No.-

The effects of metallothionein and cadmium ton on the hepatic aldehyde oxidase activity and brain lipid peroxidation were tested in vitro. The content of brain lipid peroxide at the condition of normal or 300μM Fe(Ⅱ)-induced was remarkably reduced by the addition of metallothionein in the incubation mixture. The induced content of lipid peroxide by cadmium (30μg/ml) lon was reduced by metallothionein (100μg/ml). The activity of aldehyde oxidase was not affected by metallothionein, but cadmium lon (8.38μg/ml) in creased the activity of aldehyde oxidase about 80% compared to the control. The cadmium-induced activity of aldehyde oxidase was restored to the control level by metallothionein or peniclilamine.

카드뮴을 처리한 흰쥐 간에서 Metallothionein의 방어 역할에 관한 연구

등영건,김동희,김완종 연세대학교 원주의과대학 1994 원주의대논문집 Vol.7 No.1

메탈로티오네인 단백질의 약학적 효능과 이의 응용

유한진,이지현,임광석 한국생물공학회 2019 KSBB Journal Vol.34 No.4

Metallothionein (MT) protein consists of about 60 amino acids including 20 cysteine residues. MT as a lowmolecular weight intra-cellular protein is over-expressed in the cell exposed stressful conditions such as in the presence of heavy metals, starvation, heat, or inflammation. Recently, MT has been reported as an antioxidant proteins having an antioxidant effect to protect cells from oxidative stress by reactive oxygen species (ROS). Oxidative stress has been known as a cause of various diseases such as diabetes, diabetic complications and myocardial disease and has been also found to be associated with cancer. MT has potential as a therapeutic agent due to its excellent antioxidant effect and control of cell growth. Recombinant fusion metallothionein, which is fused a protein transduction domain or mitochondrial targeted domain into metallothionein, showed enhanced therapeutic effect. In addition, the expression of metallothionein was related anticancer drug sensitivity in the cancer cells and resistance. Because MT could control cell growth and survival, it is necessary to develop a therapeutic agent targeting MT. Therefore, we discussed the characteristics and therapeutic efficacy of metallothionein.

카드뮴폭로에 대한 생물학적 지표 : Competitive ELISA에 의한 Metallothionein 정량 Assay of Metallothionein by Competitive ELiSA

박정덕,이우석,공윤,홍연표,장임원 大韓産業醫學會 1996 대한직업환경의학회지 Vol.8 No.2

Metallothionein (MT) is a cadmium binding protein that played major roles in protective mechanism for cadmium toxicity. In the present study, competitive ELISA was established to assay the MT expression utilizing monospecific antibody which was generated against MT-I. A total of 10 Sprague-Dawley rats was injected with CdCl₂ for two weeks to induce MT. The cytosolic fraction of rat liver was obtained by differential centrifugation. Two major MT isozymes (MT-I & MT-II) at ca. 10 kDa were purified by Sephadex G-75 gel filtration followed by DEAE-Trisacryl-M anion exchange column chromatography, respectively. Two rabbits were immunized 4 times consecutively with the conjugate of purified MT-I. The sera were collected by heart puncture. When immunoblot was carried out, the immunized rabbit sera (anti-MT-I) exhibited specific immunoreactive band at MT-I while showed any cross reactions neither with MT-II nor with other cytosolic proteins. By chequerboard titration using the monospecific antibody, the competitive ELISA was established. The dose-dependent relationship was observed between anti-MT-I anti-body and the amount of MT in biological samples (r²=0.9980). These results suggested strongly that competitive ELISA is a simple, rapid and reproducible method for screening cadmium exposure.

The Effect of Hydrogen Peroxide-Treated Metallothionein on the Hepatic Xanthine Oxidase Activity

Huh, Keun,Shin, Uk Seob,Lee, Sang Il 영남대학교 약품개발연구소 1996 영남대학교 약품개발연구소 연구업적집 Vol.6 No.-

We investigated the effect of hydrogen peroxide-treated metallothionein on the hepatic xanthine oxidase activity in vitro. When the metallothionein was preincubated with 1mM of hydrogen peroxide, the activity of xanthine oxidase and type conversion were elevated dose-dependently by the addition of metallothionein into the reaction mixture. While increasing the treatment of hydrogen peroxide to the 50μg of metallothionein, the xanthine oxidase activity and type conversion ratio were remarkably elevated dose dependently compared to the control. When cadmium ion was added to the reaction mixture, the increasing pattern of the enzyme activity was similar to the effect of hydrogen peroxide-treated metaliothionein. DTT or penicillamine restored the increasing activity and type conversion of xanthine oxidase by the cadmium ion to the control level.