• 제목/요약/키워드: Fluorescence polarization

검색결과 58건 처리시간 0.021초

Effects of Ionic Strength in the Medium on Sample Preconcentration Utilizing Nano-interstices between Self-Assembled Monolayers of Gold Nanoparticles

  • Nguyen, Ngoc-Viet;Wu, Jian-Sheng;Jen, Chun-Ping
    • BioChip Journal
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    • 제12권4호
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    • pp.317-325
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    • 2018
  • This paper investigated the effects of ionic strength in the medium on a preconcentrator for a protein sample with low concentration. The preconcentration chip was designed and fabricated using a polydimethylsiloxane replica through standard lithophotography. A glass substrate is silanized prior to functionalizing the nanoparticles for self-assembly at a designed region. Due to the overlap of electrical double layers in a nanofluidic channel, a concentration polarization effect can be achieved using an electric field. A nonlinear electrokinetic flow is induced, resulting in the fast accumulation of proteins in front of the induced ionic depletion zone, so called exclusion-enrichment effect. Thus, the protein sample can be driven by electroosmotic flow and accumulated at a specific location. The chip is used to collect fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) diluted in phosphate-buffered saline (PBS) buffer solution. Different concentrations of the buffer media were studied herein. Fluorescence intensity images show that the buffer concentration of 4 mM is more appropriate than all the other ones. The sample of FITC-BSA with an initial concentration of $10{\mu}M$ in the 4 mM PBS solution increases its concentration at the desired region by up to 50 times within 30 min, demonstrating the results in this investigation.

Effects of Chlorhexidine digluconate on Rotational Rate of n-(9-Anthroyloxy)stearic acid in Model Membranes of Total Lipids Extracted from Porphyromonas gingivalis Outer Membranes

  • Jang, Hye-Ock;Kim, Dong-Won;Kim, Byeong-Ill;Sim, Hong-Gu;Lee, Young-Ho;Lee, Jong-Hwa;Bae, Jung-Ha;Bae, Moon-Kyoung;Kwon, Tae-Hyuk;Yun, Il
    • The Korean Journal of Physiology and Pharmacology
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    • 제8권2호
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    • pp.83-88
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    • 2004
  • The purpose of this study was to provide a basis for studying the molecular mechanism of pharmacological action of chlorhexidine digluconate. Large unilamellar vesicles (OPGTL) were prepared with total lipids extracted from cultured Porphyromonas gingivalis outer membranes (OPG). The anthroyloxy probes were located at a graded series of depths inside a membrane, depending on its substitution position (n) in the aliphatic chain. Fluorescence polarization of n-(9-anthroyloxy)stearic acid was used to examine effects of chlorhexidine digluconate on differential rotational mobility, while changing the probes' substitution position (n) in the membrane phospholipids aliphatic chain. Magnitude of the rotational mobility of the intact six membrane components differed depending on the substitution position in the descending order of 16-(9-anthroyloxy)palmitic acid (16-AP), 12, 9, 6, 3 and 2-(9-anthroyloxy)stearic acid (12-AS, 9-AS, 6-AS, 3-AS and 2-AS). Chlorhexidine digluconate increased in a dose-dependent manner the rate of rotational mobility of hydrocarbon interior of the OPGTL prepared with total lipids extracted from cultured OPG, but decreased the mobility of membrane interface of the OPGTL. Disordering or ordering effects of chlorhexidine digluconate on membrane lipids may be responsible for some, but not all of its bacteriostatic and bactericidal actions.

Ethanol이 배양된 Mouse Myeloma Cell Line Sp2/0-Ag14로부터 분리한 형질막의 유동성에 미치는 영향 (Effects of Ethanol on the Fluidity of Plasma Membrane Vesicles Isolated from Cultured Mouse Myeloma Cell Line Sp2/0-Ag14)

  • 윤일;정인교;박영민;김진범;유성호;강정숙
    • 대한약리학회지
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    • 제29권1호
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    • pp.149-156
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    • 1993
  • Ethanol이 암세포 증식 속도에 미치는 직접적인 영향 검색의 일환으로 배양된 mouse myeloma cell line Sp2/0-Ag14로부터 분리한 형질막 (Sp2/0-PMV) 유동성에 미치는 ethanol의 영향을 형광분석법으로 측정하였다. 그 결과 ethanol은 Sp2/0-PMV 지질 이중층 측방확산운동의 범위와 속도를 증가시켰고 회전확산운동 범위도 증가시켰다. 특히 ethanol은 Sp2/0-PMV 지질이중층 중 내부단층 (inner monolayer)에 비하여 비교적 선택적으로 외부단층 (outer monolayer)의 회전확산운동 범위를 증가시킨다는 것을 확인하였다.

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The Effect of Tetracaine.HCl on Rotational Mobility of n-(9-Anthroyloxy) Stearic Acid in Outer Monolayers of Neuronal and Model Membranes

  • Joo, Hyung-Jin;Ryu, Jong-Hyo;Park, Chin-U;Jung, Sun-Il;Cha, Yun-Seok;Park, Sang-Young;Park, Jung-Un;Kwon, Soon-Gun;Bae, Moon-Kyung;Bae, Soo-Kyoung;Jang, Hye-Ock;Yun, Il
    • International Journal of Oral Biology
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    • 제35권4호
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    • pp.159-167
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    • 2010
  • To provide a basis for studying the pharmacological actions of tetracaine HCl, we analyzed the membrane activities of this local anesthetic. The n-(9-anthroyloxy) stearic and palmitic acid (n-AS) probes (n = 2, 6, 9, 12 and 16) have been used previously to examine fluorescence polarization gradients. These probes can report the environment at a graded series of depths from the surface to the center of the membrane bilayer structure. In a dosedependent manner, tetracaine HCl decreased the anisotropies of 6-AS, 9-AS, 12-AS and 16-AP in the hydrocarbon interior of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV), and liposomes derived from total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. However, this compound increased the anisotropy of 2-AS at the membrane interface. The magnitude of the membrane rotational mobility reflects the carbon atom numbers of the phospholipids comprising SPMV, SPMVTL and SPMVPL and was in the order of the 16, 12, 9, 6, and 2 positions of the aliphatic chains. The sensitivity of the effects of tetracaine HCl on the rotational mobility of the hydrocarbon interior or surface region was dependent on the carbon atom numbers in the descending order 16-AP, 12-AS, 9-AS, 6-AS and 2-AS and on whether neuronal or model membranes were involved in the descending order SPMV, SPMVPL and SPMVTL.

광합성 에너지 수용색소 분자의 Excited States (第1報) Peridinin (Excited States of Photoreceptor Molecules (I). Peridinin)

  • 송필순;이태영
    • 대한화학회지
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    • 제23권5호
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    • pp.314-319
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    • 1979
  • 바닥 말무리 dinoflagellate의 광합성 에너지 수용 색소인 peridinin의 전자 흡수스펙트럼을 PPP SCF MO 계산결과와 형광 편광 실험결과에 견주어 분석하였다. 청색광 부위의 흡수띠 (470 nm)는 $B{\leftarrow}A$ 천이에 대응하여 분자의 장축과 거의 평행 방향으로 편광되어 있음이 예측된다. 소위 "cis peak" 영역의 근자외선 흡수띠는 두개의 비교적 약한 ${\pi}{\rightarrow}{\pi}^*$천이 $(C{\leftarrow}A$$D{\leftarrow}A)$에 기인하며 그 편광축은 $^1B{\leftarrow}A$편광축 방향과 근접되어 있음이 MO 계산과 편광 측정에 의해 추정되었다. 그리고 락톤 카르보닐 원자단의 전자구조는 $^1B$ 상태에서도$^1A$ 상태 (ground state)에 비해서 과히 변화함이 없으나, allene기는 charge transfer적 성격을 농후하게 띠고 있어서 들뜬상태에서는 allene기가 전자결핍상태가 되는 것이 예측된다.

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Different Effects of Dopamine on Differential Rotational Mobility between Inner and Outer Monolayer of Synaptosomal Plasma Membrane Vesicles Isolated from Bovine Brain

  • Kim, Hyun-Gang;Choi, Chang-Hwa;Kim, Inn-Se;Chung, In-Kyo;Cho, Goon-Jae;Jang, Hye-Ock;Yun, Il
    • The Korean Journal of Physiology and Pharmacology
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    • 제4권5호
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    • pp.409-415
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    • 2000
  • Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to evaluate the effects of dopamine HCl on the range of the rotatioanl mobility of bulk bilayer structure of the synaptosomal plasma membrane vesicles (SPMV) isolated from whole bovine brain. In a dose-dependent manner, dopamine decreased the anisotropy $({\gamma}),$ limiting anisotropy $({\gamma}{infty})$ and order parameter (S) of DPH in the membranes. These indicate that dopamine increased the rotational mobility of the probe in the neuronal membranes. Cationic 1-[4-(trimethylammonio)-phenyl]-6-phenylhexa-1,3,5-hexatriene (TMA-DPH) and anionic 3-[p-(6-phenyl)-1,3,5-hexatrienyl]-phenylpropionic acid (PRO-DPH) were utilized to examine the range of transbilayer asymmetric rotational mobility of the neuronal membranes. Dopamine had a greater increasing effect on the mobility of the inner monolayer as compared to the outer monolayer of the neuronal membranes. It has been proven that dopamine exhibits a selective rather than nonselective fluidizing effect within the transbilayer domains of the SPMV.

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Overexpression of Semaphorin4D Indicates Poor Prognosis and Prompts Monocyte Differentiation toward M2 Macrophages in Epithelial Ovarian Cancer

  • Chen, Ying;Zhang, Lei;Lv, Rui;Zhang, Wen-Qi
    • Asian Pacific Journal of Cancer Prevention
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    • 제14권10호
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    • pp.5883-5890
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    • 2013
  • Previously, we demonstrated overexpression of semaphorin4D (SEMA4D, CD100) to be closely related to tumor angiogenesis in epithelial ovarian cancers (EOCs). However, the function and expression of SEMA4D in the EOC microenvironment has yet to be clarified in detail. In this study, we confirmed that overexpression of SEMA4D in primary tumors and ascites was related to low differentiation, platinum resistance and a refractory status (P<0.05), while high M2 macrophage count and percentage were evident in EOC patients with advanced FIGO stage and platinum resistance (P<0.05), using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorting (FACS), respectively. The data showed correlations of SEMA4D expression and M2 macrophage counts in primary tumors and M2 macrophage percentage in ascites (r=0.281 and 0.355, each P<0.05). In the Cox proportional hazard mode, SEMA4D expression was an independent indicator of overall survival (OS) and progression-free survival (PFS) for EOC patients. Furthermore, higher expression of SEMA4D in ovarian cancer cell lines (SKOV3, A2780, and SW626) and their supernatants were found than that in a human primary cultured ovarian cell and its supernatant by reversed transcript PCR (RT-PCR), Western blotting and ELISA, respectively. Interestingly, peripheral blood monocytes (MOs) tended towards the M2-polarized macrophage phenotype ($CD163^{high}$) in vitro after human recombined soluble SEMA4D protein stimulation. These findings suggest that SEMA4D might possibly serve as a reliable tool for early and accurate prediction of EOC poor prognosis and could playan important role in promoting tumor dissemination and metastasis in the EOC microenvironment. Thus SEMA4D and its role in macrophage polarization in EOC warrants further study.

Effects of Local Anesthetics on Rotational Mobility of n-(9-Anthroyloxy)stearic Acid in Neuronal Membranes

  • Jang, Hye-Ock;Lee, Chang;Choi, Min-Gak;Shin, Sang-Hun;Chung, In-Kyo;Yun, Il
    • The Korean Journal of Physiology and Pharmacology
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    • 제7권3호
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    • pp.119-124
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    • 2003
  • To elucidate the molecular mechanism of pharmacological action of local anesthetics, we studied membrane actions of tetracaine, bupivacaine, lidocaine, prilocaine and procaine. Fluorescence polarization of n-(9-anthroyloxy)stearic acid (n-AS) was used to examine the effects of these local anesthetics on differential rotational mobility of different positions of the number of synaptosomal plasma membrane vesicle (SPMV) phospholipid carbon atoms. The four membrane components differed with respect to 3, 6, 9 and 16-(9-anthroyloxy)stearic acid (3-AS, 6-AS, 9-AS and 16-AP) probes, indicating that differences in the membrane fluidity might be present. Degrees of the rotational mobility of 3-AS, 6-AS, 9-AS and 16-AP were different depending on depth of hydrocarbon interior. In a dose-dependentmanner, tetracaine, bupivacaine, lidocaine, prilocaine and procaine decreased anisotropy of 3-AS, 6-AS, 9-AS and 16-AP in the hydrocarbon interior of the SPMV. These results indicate that local anesthetics have significant disordering effects on hydrocarbon interior of the SPMV, thus affecting the transport of $Na^+$ and $K^+$ in nerve membranes and leading to anesthetic action.

Expression and Purification of Human Farnesoid X Receptor-Ligand Binding Domain as Soluble Form Using a Dual Cistronic Expression Vector

  • Kang, Hyun;Ye, Micheal B.;Bahk, Young Yil
    • Journal of Microbiology and Biotechnology
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    • 제23권3호
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    • pp.322-328
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    • 2013
  • In this study, we show the expression and purification of the human recombinant farnesoid X receptor (FXR)- ligand binding domain (LBD) protein in E. coli using a double cistronic vector, pACYCDuet-1, as a soluble form. We describe here the expression and characterization of a biologically active $FXR-LBD_{(248-476)}$. When expressed in the influence of bacterial promoters ($P_{T7}$ and $P_{Tac}$) of the single cistronic expression vectors, the human recombinant $FXR-LBD_{(248-476)}$ was found to be totally insoluble. However, by using a double cistronic expression vector, we were able to obtain the human recombinant $FXR-LBD_{(248-476)}$ in a soluble form. To allow for biological activities, we have subcloned into the pACYCDuet-1 vector, expressed in E. coli cells at some optimized conditions, and purified and characterized the human recombinant active $FXR-LBD_{(248-476)}$ proteins using the fluorescence polarization assay. This suggests that the expression of FXR-LBD in a double cistronic vector improves its solubility and probably assists its correct folding for the biologically active form of the proteins. We suggest that this may represent a new approach to high expression of other nuclear receptors and may be useful as well for other classes of heterodimeric protein partners.

Pharmacokinetics of Two Cyclosporine Formulations Using FPIA and HPLC Assay in Volunterrs

  • Kwon, Kwang-Il;Kim, Moo-Heon;Park, Jong-Woo;Lee, Chang-Hyun
    • Archives of Pharmacal Research
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    • 제18권6호
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    • pp.385-390
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    • 1995
  • The analytical methods for the analysis of cyclosporine (CsA), a fluorescence polarization immunoassay (FPIA) and HPLC method, were compared in a pharmacokinetic study of two CsA soft capsule formultaions ($Sandimmun^{\circledR}$; Sandoz, $Implanta^{\circledR}$; Hanmi). Sixteen healthy volunteers completed the study and each subjected single doses ($4{\tiems}100$ mg) of the test and the reference formulations in a two-way crossover design with a one-week drug-free interval between doses. Following each administration, whole blood concentrations of CsA were monitored over a period of 24 hour by both FPIA and HPLC methods. Blood concentrations nad pharmacokinetic parameters determined by either analytical method showed large intersubject variation, with the FPIA data showing relatively higher magnitude of intersubjecte variation than the HPLC data. The blood concentrations determined by FPIA were 1.1-1.3 times higher than those determined by HPLC. There were strong and significant correlations between the two methods (r>0.83 : p<0.0001). Intersubuject variation for the $AUC_{inf}{\;}and{\;}AUC_{24hr}$ of the test formulation was slightly reduced without statistical significance (paried -t test : p>0.05 $t_{max}$ was earlier nad $C_{max}$ was slightly lower for the test formulation, $AUC_{24h}, {\;}C_{max}, {\;}T_{max}$ and MRT determined separately from the data obtained by the two methods for the two formulations were examined by analyses of variance (ANOVA) for the bioequivalency evaluation. Results of ANOVA and confidence limits of terst/reference ratios of $AUC_{24th}$, $C_{max}$, $t_{max}$ and MRT, and statistical tests indicated the bioequivalence of the two formulations (i.e., test/reference ratio was within $100{\times}20%$) except for $C_{max}$ and $t_{max}$. The mean of tmax also showed 11.1% and 9.3% differences but the detection limit were 29.2% and 29.6% as determined by FPIA and HPLC resepctively. This experiments suggest that the data yielded for the two formulations demonstrated that they were bioequivalent.

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