• Journal of Plant Biotechnology
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• 제31권2호
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• pp.175-178
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• 2004

Collagen에 의해 유도되는 류마티스 관절염을 저해하는 효과가 있는 인간 histone 단백질 Hl.5 (hHl.5)를 산화스트레스 유도성 SWPA2 프로모터에 연결하여 형질전환 담배(Nicotiana tabacum L. cv Bright Yellow-2) 배양세포주를 개발하였다. hH1.5 유전자는 Agrobacterium 매개 형질전환 방법으로 담배 BY-2 배양세포에 도입되었다. 형질전환 캘러스는 150mg/L kanamycin과 300mg/L claforan이 포함된 변형된 MS 선발배지에서 선발하여, PCR분석으로 hHl.5 유전자의 도입을 확인하였다. 형질전환 현탁배양세포에서 hH1.5 단백질의 발현은 northern 분석과 Western 분석으로 확인하였는데, 담배배양세포에서 재조합hHl.5 단백질 (42 kDa)은 인간의 것 (32 kDa)과는 다른 크기의 단백질이 확인되었다. 금후 재조합 hH1.5 단백질의 자세한 특성규명이 요구된다.

• 약학회지
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• 제45권4호
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• pp.378-386
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• 2001

A purified histone Hl protein, p961, which plays a role in mediating the condensation of DNA into chromatin, was recently proved as an arthritis-suppressing agent in the mouse CIA model. The pharmacokinetics of p961 was carried out in rats and rabbits. The rat's blood, bile and urine samples were serially collected from the femoral vein, common bile duct, and bladder respectively, after bolus i.v. injection at low (10 mg/kg) and high (30 mg/mg) doses. The rabbit's blood samples were also collected from the marginal ear vein after bolus i.v. injection at a dose 10 mg/kg. p961 and its major metabolite in the physiological samples were analyzed by reverse-phase HPLC using a Yydac C4 protein column and a multistep water-acetonitrile gradient containing 0.24% trifluoroacetic acid. The major pharmacokinetic parameters (AUC, $C_{max}$, MRT, $t_{1}$2/, $V_{ss}$ and Cl) were estimated from the time course of plasma p961 and metabolite concentrations using WinNonlin. A two-compartment model was chosen for p961 as the most appropriate pharmacokinetic model. After i.v. injection of p961 at doses of 10 mg/kg and 30 mg/kg, more than 80% of p961 was removed rapidly from the plasma within 15 min. The plasma half-life of p961 in rats and rabbits was found not to exceed 12 min. p961 (22448.9 mol wt) was rapidly cleaved to 21612 mot wt fragment and the breakdown product appeared rapidly in the circulation with no lag phase. p961 and metabolite were not detected in rat urine and bile....

• Asian Pacific Journal of Cancer Prevention
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• 제15권2호
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• pp.629-635
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• 2014

Background: Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Myeloid cell leukemia-1 (Mcl-1), a death-inhibiting protein that regulates apoptosis, has been shown to be overexpressed in numerous malignancies. In addition, it has been demonstrated that the expression level of the Mcl-1 gene increases at the time of leukemic relapse following chemotherapy. The aim of this study was to target Mcl-1 by small interference RNA (siRNA) and analyze its effects on survival and chemosensitivity of acute myeloid leukemia cell line HL-60. Materials and Methods: siRNA transfection was performed with a liposome approach. The expression levels of mRNA and protein were measured by real-time quantitative PCR and Western blot analysis, respectively. Trypan blue assays were performed to evaluate tumor cell growth after siRNA transfection. The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Apoptosis was quantified using a DNA-histone ELISA assay. Results: Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a time-dependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Surprisingly, pretreatment with siMcl-1 synergistically enhanced the cytotoxic effect of etoposide. Furthermore, Mcl-1 down-regulation significantly increased apoptosis sensitivity to etoposide. No significant biological effects were observed with negative control siRNA treatment. Conclusions: Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. Therefore, siMcl-1 may be a potent adjuvant in leukemia chemotherapy.

• KSBB Journal
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• 제7권4호
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• pp.302-307
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• 1992

protein phosphatase 2A는 bovine brain homogenate의 세포질 fraction에서 얻어졌다. 기질로서 인산화된 histione H1을 이용하여 측정한 phosphatase 의 활성은 dipalmitoyIphophatidylcholine(DPPC) 혹은 phosphatidylserine/DPPC의 혼합물로 구성된 liposome의 존재하에서 저해되었다. Protein phosphatase 2A의 lipid membrane에의 결합은 다중층 지질막의 혼합물 계에서 liposome 의 양이 증가함에 따라서 상등액 중의 phosphatase의 활성이 감소하는 것으로 확인할 수 있었다. 또한 [$^{125}I$]protein phosphatase 2A가 liposome과 동시에 용출되는 것으로도 확인되었다. 그러나 liposome에 대한 protein phosphatase의 친화력은 높지 않았다. 한편, okadaic acid와 liposome은 협동으로 phosphatase의 활성을 감소시켰다. 이것은 okadaic acid가 lipid membrane이나 membrane에 결함한 phosphatase에는 결합하지 않는다는 것을 의미한다. 그러므로 lipid membrane에 의한 protein phosphatase 2A의 활성 저해 효과는 phosphatase 2A와 lipid membrane과의 결합에 의한 것이라고 설명될 수있다.

• 한국동물학회지
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• 제35권3호
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• pp.277-286
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• 1992

개구리의 난자로 부터 maturation promoting factor(MPF)를 추출, 부분 분리하여 이들의 활성을 조사하고 이 물질의 생성과 protein kinase C(반KC)와의 관계를 조사하SB다. 성숙된 난자를 분쇄한 후 초원심분리과정을 거쳐 MPF의 crude extract(CE)를 얻은 다음 ultrafiltration (UF)과 고속액체크로마토그라피를 거쳐서 3종류의 분획 (peak 1, 11, and 111)을 얻었다. 이들 분획을 in nitro assay와 autoradiDgraphy를 사용하여 확인한 결과 분획 11에서 MPF 활성이 있는 것을 알았다. 분리 단계에 따라 MPF의 정제도를 Hl histone kinase assay로 조사한 결깍 UF를 거친 것은 CE보다 약 3배로, 분획 11에서는 약 117배로 증가한 것을 확인하였다. 또한 MPF분획의 인산화를 autoradiography로 조사한 결과 45 KD 단백질을 포함한 수종의 난자 단백질이 강하게 인산화되었음을 알 수 있었다. PKC의 활성화가 난자내 MPF의 생성을 유도하는가를 보기 위하여 PKC의 활성제인 12-0-tetradecanoyl phorbol 13 acetate(TPA)를 처리한 난자의 세포질 추출물을 미세주입 법으로 조사한 결과 TPA 처리 후 6시간부터 난자내 MPF의 활성이 나타나는 것을 알 수 있었다. 이러한 결과들은 PKC의 활성화가 MPF의 생성을 유도하고, MPF의 활성화와 함께 일부 단백질들의 인산화를 통하여 궁극적으로 난자 성숙을 촉진했음을 시사한다.

• Biomolecules & Therapeutics
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• 제29권1호
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• pp.73-82
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• 2021

Hsp90 is often overexpressed with activated form in cancer cells, and many key cellular proteins are dependent upon the Hsp90 machinery (these proteins are called "client protein"). Nowadays, more client proteins and more inhibitors of Hsp90 are being discovered. Chaetocin has been identified as an inhibitor of histone methyl transferase SUV39H1. Herein, we find that Chaetocin is an inhibitor of Hsp90 which binds to the C-terminal of Hsp90α. Chaetocin inhibited a variety of Hsp90 client proteins including AMl1-ETO and BCL-ABL, the mutant fusion-protein in the K562 and HL-60 cells. SUV39H1 mediates epigenetic events in the pathophysiology of hematopoietic disorders. We found that inhibition of Hsp90 by Chaetocin and 17-AAG had ability to induce degradation of SUV39H1 through proteasome pathway. In addition, SUV39H1 interacted with Hsp90 through co-chaperone HOP. These results suggest that SUV39H1 belongs to a client protein of Hsp90. Moreover, Chaetocin was able to induce cell differentiation in the two cells in the concentration range of Hsp90 inhibition. Altogether, our results demonstrate that SUV39H1 is a new client protein of Hsp90 degradated by Chaetocin as a novel C-terminal inhibitor of Hsp90. The study establishes a new relationship of Chaetocin and SUV39H1, and paves an avenue for exploring a new strategy to target SUV39H1 by inhibition of Hsp90 in leukemia.

• Journal of Microbiology and Biotechnology
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• 제12권4호
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• pp.592-597
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• 2002

The Rkp1/CPC2, a receptor for activated protein kinase C of Schizosaccharomyces pombe, contains seven WD motifs found in the G-protein $\beta$-subunit. A 110-kDa protein was identified to interact with Rkp1/CPC2 by immunoprecipitation and following in vitro binding assay. To examine its kinase activity and binding ability to Rkp1, the $pck1^+$, a PKC homolog of S. pombe, was cloned. Pckl phosphorylated myelin basic protein (MBP) and histone Hl in a phospholipid-dependent and $Ca^{2+}$-independent manner. It was demonstrated that the N-terminal region of Pck1 was responsible for the binding to Rkp1 , thus suggesting that Rkp1 interacted with Pckl to regulate Pckl-mediated signaling in S. pombe.