• 생명과학회지
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• 제16권1호
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• pp.131-135
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• 2006

미생물이 이용할 수 있는 nitrogen source는 di-, tri-, oli- go-peptide 또는 amino acid의 형태로 세포내로 uptake되어 대사과정에 사용되고 있다. 이와같은 peptide는 특이한 transport system에 의해서 이동되고 있는데 oligo peptide(Opp) transport system에는 binding protein, permease protein, energy 생성을 위한 ATP 분해에 관여하는 protein 이 관여하고 있으며 염색체 상에서 이들 단백질들은 operon 형태의 유전자로부터 발현되고 있다. 본 연구는 gram 음성 세균이며 수해양 서식 세균인 V, fluvialis로부터 얻어진 Opp operon 유전자 가운데 oligopeptide binding protein을 coding하고 있는 oppA 유전자가 deletion된 mutant를 사용하여 여러 환경변화에 따른 생육을 wild type과 비교한 연구 결과 이다. 생육을 위한 완전배지인 brain heart infusion (BHI) 배지와 최소배지인 M9 minimal 배지를 사용한 결과 OppA protein의 생성 결핍에 따라 초기 및 대수증식기 과정 중에는 mutant의 생육이 늦어지고 있으나 Opp system이 아닌 다른 peptide전달 경로로 추정되는 system을 이용하여 대수 증식기 후반에서는 wild type과 거의 같은 생육 형태를 보여 주고 있었다. pH의 변화에 따른 생육은 pH 7에서는 생육정도가 비슷하였으나 약알칼리 부근에서는 oppA mutant의 생육이 wild type에 비하여 낮아지고 있었다. 또한 5 mM $H_2O_2$를 사용하여 $OD_{600}=1.2$농도의 세포들에 대한 영향을 검토한 결과 두 균 모두 높은 생존율을 보여 주었으며 이는 대수증식기 세포들을 사용한 결과와는 매우 다른 형태를 보여 주고 있었다. 항생제 내성에 대한 연구에서는 mutant가 streptomycin과 tetracycline 에 대해서는 wild type과는 다르게 매우 낮은 농도에서도 생육이 되고 있지 않으나 polymyxin B에 대해서는 wild type과 같이 $10{\mu}g/ml$의 농도에서도 잘 자라고 있었다.

• 대한한방종양학회지
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• 제6권1호
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• pp.81-97
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• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.

• The Korean Journal of Physiology
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• 제8권2호
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• pp.67-74
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• 1974

The object of this research is aimed to determine the activity of adenyl cyclase in both skeletal muscle sarcolemma and fat cell ghost of epididymal adipose tissue isolated from rats exposed to cold for various length of time in an attempt to evaluate whether the tissue sensitivity to catecholamine is increased when rats are exposed to cold for long periods of time Methods: a)Animals: Albino rats ranging in weight from 150 to 200 gm were used throughout this study. For experimental purposes, the rats are divided into two groups: experimental animals were place4 in a cold room at $4^{\circ}C$, controls being kept at $25^{\circ}C$. At the end of 2, 4, 6, 12, and 16 weeks. exposure to cold the rats were used to measure the adenyl cyclase activity. b) Isolation of plasma membrane from skeletal muscle and adipose tissue: The Plasma membrane of skeletal muscle from hind limbs of rats are prepared by the method employed by Rosenthal et at. and fat cell ghost of epididymal adipose tissue of rats by the method employed by Rodbell. c) Adenyl cyclase assay: Adenyl cyclase activity were measured by the method employed by Marinetti et al. Briefly, plasma membrane was incubated with $3^H-ATP$, various amount of noradrenaline and other incubation mixture at $37^{\circ}C$ for 20 minutes. After stopping the enzyme reaction by immersion in boiling water, carrier 3',5'-AMP was added to the system as a marker and $100\;{\mu}1$ aliquots of incubation mixture were pipetted on $20{\time}20$ Whatman No. 3 MM filter paper for one dimensional chromatography. The cyclic AMP spots were cut off and placed in counting vials containing 10ml of Bray's scintillation cocktail. Radioactivity was determined with a Packard Tri-Carb liquid scintillation counter. The enzyme activity is expressed as nanomoles of cyclic AMP produced per mg of membrane per hour. Result: 1. Average adenyl cyclase activity in the plasma membrane of skeletal muscle before and after noradrenaline administration was significantly higher in the cold-exposed rats as compared to the control. Continuous exposure to cold Produced an increased adenyl cyclase activity before and after noradrenaline administration. Adenyl cyclase activity reached peak levels at the 6 weeks exposure to told and level of adenyl cyclase activity remained high. Noradrenaline administration to the incubation medium induced a significant increase in adenyl cyclase activity and the degree of stimulation were proportional to the hormonal concentration But the rate of inclement in adenyl cyclase activity by noradreasline was the same in both groups. 2. Adenyl cyclase activity in fat cell ghost between cold exposed and control rats showed no significant differences before and after noradreualine administration. In summary, it can be concluded that cold adaptation give rise an increased activity of adenyl cyclase in plasma membrane of skeletal muscle in rats.

• Journal of Microbiology and Biotechnology
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• 제32권9호
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• pp.1154-1167
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• 2022

In this study, we investigated the anti-amnesic effect of Korean red pine (Pinus densiflora) bark extract (KRPBE) against amyloid beta_1-42 (Aβ_1-42)-induced neurotoxicity. We found that treatment with KRPBE improved the behavioral function in Aβ-induced mice, and also boosted the antioxidant system in mice by decreasing malondialdehyde (MDA) content, increasing superoxide dismutase (SOD) activities, and reducing glutathione (GSH) levels. In addition, KRPBE improved the cholinergic system by suppressing reduced acetylcholine (ACh) content while also activating acetylcholinesterase (AChE), regulating the expression of choline acetyltransferase (ChAT), postsynaptic density protein-95 (PSD-95), and synaptophysin. KRPBE also showed an ameliorating effect on cerebral mitochondrial deficit by regulating reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and ATP levels. Moreover, KRPBE modulated the expression levels of neurotoxicity indicators Aβ and phosphorylated tau (p-tau) and inflammatory cytokines TNF-α, p-IκB-α, and IL-1β. Furthermore, we found that KRPBE improved the expression levels of neuronal apoptosis-related markers BAX and BCl-2 and increased the expression levels of BDNF and p-CREB. Therefore, this study suggests that KRPBE treatment has an anti-amnestic effect by modulating cholinergic system dysfunction and neuroinflammation in Aβ_1-42-induced cognitive impairment in mice.

• Archives of Pharmacal Research
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• 제29권3호
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• pp.224-234
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• 2006

We employed human SK-MEL-28 cells as a model system to identify cellular proteins that accompany N-(4-methyl)phenyl-O-(4-methoxy)phenyl-thionocarbamate (MMTC)-induced apoptosis based on a proteomic approach. Cell viability tests revealed that SK-MEL-28 skin cancer cells underwent more cell death than normal HaCaT cells in a dose-dependent manner after treatment with MMTC. Two-dimensional electrophoresis in conjunction with matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis or computer matching with a protein database further revealed that the MMTC-induced apoptosis is accompanied by increased levels of caspase-1, checkpoint suppressor-1, caspase-4, NF-kB inhibitor, AP-2, c-Jun-N-terminal kinase, melanoma inhibitor, granzyme K, G1/S specific cyclin D3, cystein rich protein, Ras-related protein Rab-37 or Ras-related protein Rab-13, and reduced levels of EMS (oncogene), ATP synthase, tyrosine-phosphatase, Cdc25c, 14-3-3 protein or specific structure of nuclear receptor. The migration suppressing effect of MMTC on SK-MEL-28 cell was tested. MMTC suppressed the metastasis of SK-MEL-8 cells. It was also identified that MMTC had little angiogenic effect because it did not suppress the proliferation of HUVEC cell line. These results suggest that MMTC is a novel chemotherapeutic and metastatic agents against the SK-MEL-28 human melanoma cell line.

• BMB Reports
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• 제36권6호
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• pp.545-551
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• 2003

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.104-114
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• 2024

Licochalcone C (LCC; PubChem CID:9840805), a chalcone compound originating from the root of Glycyrrhiza inflata, has shown anticancer activity against skin cancer, esophageal squamous cell carcinoma, and oral squamous cell carcinoma. However, the therapeutic potential of LCC in treating colorectal cancer (CRC) and its underlying molecular mechanisms remain unclear. Chemotherapy for CRC is challenging because of the development of drug resistance. In this study, we examined the antiproliferative activity of LCC in human colorectal carcinoma HCT116 cells, oxaliplatin (Ox) sensitive and Ox-resistant HCT116 cells (HCT116-OxR). LCC significantly and selectively inhibited the growth of HCT116 and HCT116-OxR cells. An in vitro kinase assay showed that LCC inhibited the kinase activities of EGFR and AKT. Molecular docking simulations using AutoDock Vina indicated that LCC could be in ATP-binding pockets. Decreased phosphorylation of EGFR and AKT was observed in the LCC-treated cells. In addition, LCC induced cell cycle arrest by modulating the expression of cell cycle regulators p21, p27, cyclin B1, and cdc2. LCC treatment induced ROS generation in CRC cells, and the ROS induction was accompanied by the phosphorylation of JNK and p38 kinases. Moreover, LCC dysregulated mitochondrial membrane potential (MMP), and the disruption of MMP resulted in the release of cytochrome c into the cytoplasm and activation of caspases to execute apoptosis. Overall, LCC showed anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting EGFR and AKT, inducing ROS generation and disrupting MMP. Thus, LCC may be potential therapeutic agents for the treatment of Ox-resistant CRC cells.

• 한국수산과학회지
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• 제18권2호
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• pp.131-138
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• 1985

다획성적색육어류에 속하는 고등어를 효율적으로 이용하기 위한 방안의 하나로서 즉석에서 그대로 조리하여 먹을 수 있도록 알맞은 수분함양과 식염함양이 적은 반염건고등어를 제조하여 함기포장한 것을 대조제품으로 하고 탈산소제를 봉입한 포장제품을 진공포장제품, 질소치환포장제품과 비교하여 저장중의 품질안정성에 대하여 검토하였다. pH는 전제품이 $6.1{\pm}0.2$의 범위내에서 저장중 약간의 증감이 있었으며, 휘발성염기질소와 아미노질소는 전제품이 저장중 증가하는 경향이었다. 특히 탈산소제봉입포장제품은 휘발성염기질소의 증가폭이 낮았다. 색조는 저장중 전제품이 L값(명도)이 약간 감소한 반면에 a값(적색도) 및 b값(황색도)은 증가하는 경향이었다. 그리고 함기포장제품(대조제품)의 경우 생균수는 저장 15일경에 $3{\times}10^6/g$에 달하였으나 진공, 질소치환 및 탈산소제봉입포장제품은 저장20일까지도 $2{\sim}6{\times}10^5/g$범위였다. TMAO는 저장중 감소하고 TMA는 증가하는 역상관관계를 나타내었으며, histamine은 전제품 모두가 16mg/100g이 하였다. 또한 핵산관련물질은 초기에 IMP가 축적되었으나 저장기간이 경과함에 따라 IMP가 분해되고 이노신과 하이포크산틴이 축적되었다. TBA값은 함기포장제품의 경우 저장 9일째 최고값을 나타내다가 감소하였으며, 진공포장제품과 질소치환포장제품은 저장 9일째 최고값을 나타내었으나, 함기포장제품의 경우보다 1/2 정도로 낮았다. 그러나 탈산소제봉입포장제품은 저장중 거의 변화가 없었으며, 과산화물 값도 비슷한 경향이었다. 생시료는 포화지방산이 $35.6\%$, polyene 산이 $34.2\%$, monoene 산이 $30.3\%$였고 oleic acid가 $20.2\%$로 가장 많았고 다음으로 palmitic acid, docosahexaenoic acid, eicosapentaenoic acid순이였다. 저장중 포장방법에 관계없이 고도불포화지방산($C_{20:5},\;C_{22:6}$)이 감소하는 반면에 $C_{16:0},\;C_{18:1}$은 다소 증가하는 경향을 나타내었으나, 탈산소제봉입포장제품이 고도불포화지방산의 보존효과가 가장 좋았다. 관능검사 결과, 함기포장제품은 저장 9일째에 선도저하에 의한 이취로 식용할 수 없을 정도였으나, 진공포장, 질소치환포장제품 및 탈산소제봉입포장제품은 저장 15일까지도 식용가능하였다. 특히 탈산소제봉입포장제품은 진공포장제품 및 질소치환포장제품과 마찬가지로 품질보존효과가 우수하다는 결론을 얻었다.