BOXTO as a real-time thermal cycling reporter dye

The unsymmetrical cyanine dyes BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) and its positive divalent derivative BOXTO-PRO (4-[3-methyl-6-(benzoxazole-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-(3-trimethylammonium-propyl)-quinolinium dibromide) were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG) (2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium). Unmodified BOXTO showed no inhibitory effects on real-time PCR, while BOXTO-PRO showed complete inhibition, Sufficient fluorescent signal was acquired when 0.5–1.0 μM BOXTO was used with RotorGene and iCycler platforms. Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO and SG. BOXTO stock solution (1.5 mM) was stable at −20°C for more than one year and 40 μM BOXTO solution was more stable than 5x SG when both were stored at 4°C for 45 days.     

Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay

Abstract

Fluorescence spectroscopy was used to study the ability of dye 7519 to follow the transition of monomeric insulin into fibrils and applicability of the dye to the insulin aggregation inhibition assay. The commercially available classic amyloid stain, thioflavin T, was used as the reference dye. For selecting potential inhibitors, the QSAR approach was applied. Dye 7519 appeared to be suitable for monitoring insulin aggregation into fibrils in vitro. The properties of the dye allowed us to test it as a potential probe in the screening assay of potential inhibitors of insulin fibrillization. One hundred forty-four flavonoids were tested as potential inhibitors of amyloid fibril formation using the quantitative structure activity relationship approach. Among them, 10 candidates with high indexes of inhibition were selected for tests in vitro using dye 7519 and the reference amyloid dye thioflavine T. Using dye 7519 fluorescence, we found that two compounds had inhibitory effects on insulin amyloid formation. These results agree with inhibition data using the thioflavine T assay. Our studies demonstrated that the fluorescent cyanine dye 7519 is a sensitive probe for quantitative detection of insulin amyloid formation and can be applied to screen agents capable of affecting aggregation of amyloid proteins.     

bcl-2和bax及NF-kB在白藜芦醇诱导肝癌细胞凋亡中的作用

目的探讨白藜芦醇诱导肝癌细胞凋亡的途径。方法白藜芦醇体外处理HepG2肝癌细胞24h后,以免疫组化检测凋亡调控基因bc1-2和bax及NF-kB的表达。结果白藜芦醇处理组HepG2细胞bc1-2的阳性积分和NF-kB的阳性细胞密度均明显低于对照组(P<0.01);而bax阳性积分明显高于对照组(P<0.01)。结论白藜芦醇能下调HepG2细胞bc1-2基因的表达,上调bax的表达,同时抑制NF-kB的活化,这可能是其诱导HepG2细胞凋亡的途径之一。     

七甲川花菁染料作为肿瘤靶向和光动力治疗载体的研究进展

七甲川花菁近红外荧光染料(NIRF)可直接被肿瘤细胞特异性吸收,具有肿瘤靶向性。与化疗药物偶联后,该类染料可通过血脑屏障将药物转运至肿瘤部位,不仅可以减少化疗药物使用剂量,降低药物的毒副作用,也可通过近红外荧光成像实现对肿瘤治疗的实时监控。七甲川花菁染料所展示的线粒体毒性和光敏特性,可直接杀死肿瘤细胞,抑制肿瘤新生血管的形成。通过纳米包裹,能够显著增强该类染料的肿瘤靶向能力,实现实时跟踪药物释放情况。七甲川花菁染料特异性识别肿瘤细胞的能力与有机阴离子转运肽的作用密切相关,缺氧和线粒体膜电位也参与了染料吸收的调控。这些发现有利于将近红外荧光染料应用于肿瘤的靶向治疗。     

Ab initio (CASPT2) excited state calculations, including circular dichroism, of helically twisted cyanine dyes

Ab initio calculations at the CASSCF/CASPT2 level were performed on helically twisted mono-, tri-, and pentamethine cyanine dyes in the all-Z-configurations. Excitation energies and oscillator and rotatory strengths were calculated for the five lowest energy singlet states. Both the long wavelength methine band and the cis-band could be identified unambiguously from their configurational parentage. The calculated state energies are within 0.09 eV of the experimental value for the methine band and within 0.16 eV for the cis-band. The calculated rotatory strengths of the methine band shows sign inversion as the length of the chromophore increases: negative for the short monomethine, strongly positive for the pentamethine. The trimethine presents a borderline case: the measured rotatory strength is almost nil, the calculated one depends on the geometry. There is good agreement between rotatory strengths calculated in the velocity and in the length formalism.     

Synthesis and functional characterization of a fluorescent peptide probe for non invasive imaging of collagen in live tissues

Targeted molecular imaging to detect changes in the structural and functional organization of tissues, at the molecular level, is a promising approach for effective and early diagnosis of diseases. Quantitative and qualitative changes in type I collagen, which is a major component in the extra cellular matrix (ECM) of skin and other vital organs like lung, liver, heart and kidneys, are often associated with the pathophysiology of these organs. We have synthesized a fluorescent probe that comprises collagelin, a specific collagen binding peptide, coupled to fluorescent porphyrin that can effectively detect abnormal deposition of collagen in live tissues by emitting fluorescence in the near infra red (NIR) region. In this report we have presented the methodology for coupling of 5-(4-carboxy phenyl)-10, 15, 20-triphenyl porphyrin (C-TPP) to the N-terminal of collagelin or to another mutant peptide (used as a control). We have evaluated the efficacy of these fluorescent peptides to detect collagen deposition in live normal and abnormal tissues. Our results strongly suggest that porphyrin-tagged collagelin can be used as an effective probe for the non invasive in vivo detection of tissue fibrosis, especially in the liver.     

Studies of monomeric and homodimeric oxazolo[4,5-b]pyridinium cyanine dyes as fluorescent probes for nucleic acids visualization

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.     

百里香酚的抗肿瘤作用

目的：观察百里香酚对体外培养的肝癌细胞的抑制作用。方法：体外培养人肝癌细胞（Bel-7402）,采用MTT法、AO/EB荧光染色法观察百里香酚对人肝癌细胞Bel-7402的作用。结果：百里香酚可显著抑制Bel-7402细胞的生长;经百里香酚作用后,肝癌细胞在显微镜形态明显改变。结论：百里香酚能抑制肝癌Bel-7402细胞生长。     

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.     

Postcolumn nucleic acid intercalation for the fluorescent detection of nucleic acids using ion pair reverse phase high-performance liquid chromatography

Here we report a simple and effective procedure enabling the fluorescent detection of nucleic acids following the rapid, high-resolution separation using ion pair reverse phase chromatography. This approach uses postcolumn nucleic acid intercalation of fluorescent dyes with subsequent fluorescent detection, demonstrating more than a 1000-fold increase in sensitivity in the detection of nucleic acids when compared with traditional UV detection. Moreover, a wide range of intercalating dyes can be incorporated, including those known to disrupt the structure of the nucleic acids, thereby enabling the sensitive detection of DNA and RNA with no adverse effect on resolution of the nucleic acids during ion pair reverse phase chromatography. In addition, such approaches allow one to readily distinguish single-stranded DNA from double-stranded DNA following their separation using ion pair reverse phase high-performance liquid chromatography.     

HepG2及其克隆形成细胞端粒酶活性的异质性研究

目的研究端粒酶在肝癌细胞株HepG2及其克隆形成细胞中的表达,探讨不同增殖能力的肝癌细胞中端粒酶活性的异质性,为肝癌的诊断以及治疗提供新的思路。方法利用软琼脂克隆形成实验富集分离人肝癌HepG2细胞的克隆形成细胞;常规培养HepG2及其克隆形成细胞,利用免疫细胞化学、Western blotting和RT-PCR检测hTERT蛋白和mRNA在HepG2细胞及其克隆形成细胞中表达的异质性。结果①HE染色显示,克隆形成细胞的胞核较HepG2细胞大,核仁明显;细胞伸出较多的细长突起,并连接形成网状。②免疫细胞化学染色显示,hTERT在HepG2细胞的表达以细胞质为主,克隆形成细胞则以细胞核为主。③Western blotting和RT-PCR结果显示,克隆形成细胞中hTERT蛋白质和mRNA的表达均高于HepG2细胞。结论①hTERT在HepG2细胞及其克隆形成细胞中的表达存在异质性,且在克隆形成细胞中表达较高。②hTERT在HepG2细胞及其克隆形成细胞中的表达模式提示克隆形成细胞具有肝癌干细胞的特征。     

骨形成蛋白7基因诱导人肝癌细胞的凋亡

目的 构建表达重组人骨形成蛋白7 （bone morphogenic protein 7, BMP7）基因的重组逆转录病毒,观察其对人肝癌细胞HepG2的凋亡诱导活性,并探讨其作用机制。方法 克隆BMP7基因,以loxP同源重组法构成逆转录病毒载体pLP-LNCX-BMP7（pLLBMP7）,转染包装细胞PT67进行病毒包装并测定病毒滴度;将逆转录病毒感染人成骨细胞,MTT法检测细胞生长变化,琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;Western blotting检测BMP7,caspase-3和bcl-2蛋白表达。结果 重组逆转录病毒载体pLLBMP7经鉴定连接正确,转染PT67细胞后上清液中可得到病毒,滴度达1×109pfu;MTT检测见pLLBMP7病毒组48和72h细胞抑制率高于对照组（35.1% vs. 5.3%,68.5% vs.18.3%,均p<0.05）,48h可见BMP7蛋白高表达。琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高峰,其凋亡百分率高达14.42%;BMP7蛋白高表达时caspase-3蛋白的表达亦有显著升高,但bcl-2蛋白未见表达差异。结论 构建了BMP7逆转录病毒,在体外能够有效地诱导人肝癌细胞HepG2的凋亡,其可能是通过激活caspase-3而发生作用。