2-(Phenylazo)pyridineplatinum(II) Catecholates Showing Photocytotoxicity, Nuclear Uptake, and Glutathione-Triggered Ligand Release

Platinum(II) complexes Pt(pap)(an-cat)] (1) and Pt(pap)(py-cat)] (2) with 2-(phenylazo)pyridine (pap), 4-2-(anthracen-9-ylmethylene)amino]ethyl]benzene-1,2-diol (H(2)an-cat), and 4-2-(pyren-1-ylmethylene)amino]ethyl]benzene-1,2-diol (H2py-cat) were prepared, and their photoinduced cytotoxicity was studied. The complexes were found to release catecholate ligand in the presence of excess glutathione (GSH), resulting in cellular toxicity in the cancer cells. The catecholate complex Pt(pap)(cat)] (3) was prepared and used as a control. Complex 3, which is structurally characterized by X-ray crystallography, has platinum(II) in a distorted square-planar geometry. The complexes are redox-active, showing responses near 0.6 and 1.0 V versus SCE in N,N-dimethylformamide/0.1 M tetrabutylammonium perchlorate corresponding to a two-step catechol oxidation process and at -0.3 and -1.3 V for reduction of the pap ligand. Complex 1 showed remarkable light-induced cytotoxicity in HaCaT (human skin keratinocytes) and MCF-7 (human breast cancer) cells, giving IC50 value of similar to 5 mu M in visible light of 400-700 nm and >40 mu M in the dark. The 2',7'-dichlorofluorescein diacetate (DCFDA) assay showed the generation of reactive oxygen species (ROS), which seems to trigger apoptosis, as is evident from the annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. The fluorescence microscopic images showed significant nuclear localization of the complexes and free ligands. A mechanistic study revealed possible reduction of the coordinated azo bond of pap by cellular GSH, releasing the catecholate ligand and resulting in remarkable photochemotherapeutic action of the complexes.

异硫氰酸荧光素-溶菌酶的制备及晶体生长预研究

迄今尚未有适用于光源为488 nm激光扫描共聚焦显微镜研究用的溶菌酶.为此,用异硫氰酸荧光素作为探针标记了溶菌酶,测定了溶菌酶标记物的紫外-可见吸收光谱和荧光光谱,摸索了其晶体的生长条件.实验结果表明,在标记过程中异硫氰酸荧光素没有影响溶菌酶的生化性质,标记后的溶菌酶可用于激光扫描共聚焦显微镜进行后续研究.

沢吨染料的光化漂白动力学及全息特性研究

研究了以三乙醇胺作为引发剂,五种沢吨染料的光化漂白过程.实验结果表明五种染料的光化漂白速率大小顺序是藻红B（ErB）＞曙红Y（EY）＞孟加拉玫瑰红B（RsB）＞罗丹明B（RoB）＞荧光素（F）.并研究了曝光强度及染料浓度对漂白过程的影响,以及这五种染料的全息记录性能.

Estudo fenotípico da região Aorta-Gônada-Mesonefros, de embriões de galinhas Gallus gallus domesticus, com ênfase na aorta dorsal

Acredita-se que os primeiros progenitores da hematopoese definitiva surjam da diferenciação do endotélio da aorta dorsal, na altura da região da Aorta-Gônada-Mesonefros (AGM). Com o intuito de estudar esta região e o fenótipo das células do endotélio da aorta dorsal nesta posição topográfica, ovos galados de Gallus gallus domesticus L. foram incubados em chocadeira, classificados em estádios de E16 a E25 e processados histotecnologicamente para obtenção de secções seriadas na altura da região AGM. Estas passaram por coloração por Hematoxilina-Eosina, histoquímica para PAS, PAS-diastase e Alcian Blue pH 1.0 e pH 2.5, histoquímica por lectinas fluoresceinadas e imunofluorescência para moléculas de superfície, citoesqueleto e matriz extracelular. Foi observada hipertrofia endotelial no assoalho da aorta nos estádios observados, o qual se apresentava positivo ao PAS, com ocorrência frequente de vacuolizações basais PAS negativas, e o surgimento ocasional de grupamentos celulares intravasculares. Nestes, as células que se destacavam da membrana basal do endotélio expressavam progressivamente mais material PAS positivo, o qual, no entanto, em nenhum momento pareceu se tratar de glicogênio. Em relação às glicosaminoglicanas, notamos a presença predominante de ácido hialurônico por todo o mesênquima da região e em outras estruturas como periferia da notocorda, tubo neural e mesoderma lateral. Ocorreu co-expressão de fibronectina e α-actina de músculo liso em células circunjacentes à aorta, na face ventral do vaso. GFAP e BMP-4 são expressas entre as células do tubo neural e em sua periferia, assim como na notocorda do embrião. As lectinas Abrus precatorius, Lens culinarise Ricinus communis mostraram-se positivas principalmente na região subedotelial do assoalho da aorta nos estádios observados neste trabalho. Bandeiraea simplicifolia exibiu pouca marcação na aorta dorsal e a Arachis hypogeae foi negativa. Outras estruturas da região AGM também expressaram resíduos de açúcares revelados por estas lectinas, tais como: notocorda, tubo neural, mesênquima, intestino primitivo e saco vitelínico. Estes resultados acrescentam elementos morfológicos e bioquímicos ao conhecimento sobre a região AGM de embriões de galinha e sobre o endotélio, possivelmente hemogênico, da aorta dorsal.

Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry

Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UV excited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342/PI/SYBR-14 triple staining method, it was found that the live/dead sperm distributions were completely concordant in both H342/PI and SYBR-14/PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better Preservation of plasma and acrosome integrity than TEST Therefore, the H342/PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function.

Relationship between trichosanthin cytotoxicity and its intracellular concentration

Trichosanthin (TCS) is a type I ribosome-inactivating protein with board spectrum of biological activity. Toxicity of this compound differs in different cell lines and this study examined the cause of such difference. It is generally believed that TCS toxicity is mediated through intracellular ribosome inactivation. Therefore, TCS toxicity should be determined by the amount inside cells rather than outside. Three different cell types IC21, JAR and Vero cell lines were chosen with high, medium and low sensitivity to TCS. Intracellular concentrations of fluorescein isothiocyanate labeled TCS were determined by laser scanning confocal microscopy. A good relationship was demonstrated between intracellular TCS concentration and toxicity. Highest intracellular concentration was found in IC21, followed by JAR, and lowest in Vero cells. When the intracellular TCS concentrations in these cells were reduced by using a competitive inhibitor to block cell entry, cytotoxicity was not observed. In conclusion, there is strong evidence to indicate that cytotoxicity of TCS is dependent on its intracellular concentration. Variation of cytotoxicity in different cells may be related to the mechanisms affecting its internalization. (C) 2002 Published by Elsevier Science Ireland Ltd.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

Preparation of pH-sensitive polymer by thermal initiation polymerization and its application in fluorescence immunoassay

A fluorescence immunoassay for human IgG (Ag) was developed using a pH-sensitive polymer prepared by thermal initiation or redox initiation polymerization as a carrier. In the competitive immunoassay, appropriate quantity of Ag was immobilized on the polymer and the standard Ag (or sample) solution, and a constant amount of fluorescein isothiocyanate labeled goat anti-human IgG antibody (Ab-FITC) was added. Immobilized Ag and the standard (or sample) Ag competed for binding to the Ab-FITC in 37 C in homogeneous format. After changing the pH to separate the polymer-immune complex precipitate, it was re-dissolved and determined by fluorescence method. The results showed that the immobilization efficiency, immunological reaction activities of immobilized Au and phase transition pH range were improved as Ag was immobilized by thermal initiation instead of redox initiation polymerization. Under optimum conditions, the calibration graphs for the Ag in both methods, thermal initiation and redox initiation, were linear over the concentration range of 0.0-1000 ng mL(-1), with detection limits 8 (thermal initiation) and 12 ng mL(1) (redox initiation), respectively. Moreover, some pH-sensitive polymer prepared only in organic solvent or under high temperature could also be used as an immunoreaction carrier by thermal initiation polymerization. Thermal initiation polymerization was a better immobilization mode. (C) 2004 Elsevier B.V. All rights reserved.

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.

A miniaturized fluorescence detector with a windowless flow cell using a light-emitting diode

A miniaturized fluorescence detector using a high-brightness light-emitting diode as an excitation source was constructed and evaluated. A windowless flow cell based on a commercial four-port cross fitting was designed to reduce the stray-light level and to eliminate the optical alignment. The observed detection limit for fluorescein was 26 nM in the continuous-flow mode. The error in the reproducibility of the responses was evaluated by the FIA method, and was found to be within 2% RSD.

Light-emitting-diode-induced fluorescence detector for capillary electrophoresis using optical fiber with spherical end

A simple fluorescence detector for capillary electrophoresis (CE) using a blue light-emitting-diode (LED) as excitation source is constructed and evaluated. An optical fiber was used to collect the fluorescence, and a flat end of the fiber was modified to spherical end, resulting in 50% increase of efficiency over the flat end. A simple device for optical alignment of the fibers and capillary column was designed. The concentration and mass detection limits for fluorescein were 1.8 x 10(-7) Mol l(-1) and 4.3 femol, respectively. (C) 2002 Elsevier Science B.V. All rights reserved.

Capillary electrophoretic analysis of arsenic species with indirect laser induced fluorescence detection

The development of a method for determining arsenic species by capillary zone electrophoresis (CZE) with indirect laser-induced fluorescence (LIF) is described in this paper. The buffer pH, the concentration of fluorescein, the nature and the concentration of the background electrolytes (BGEs) were defined. When 2.0 mM NaHCO3 (pH 9.28) with 10(-7) M fluorescein was used as the buffer, arsenite (As(lll), dimethylarsonic acid (DMA), monomethylarsonic acid (MMA), and arsenate (As(V)) were all separated from one another. The limits of detection for the four arsenic species were p p in the range of 0.12-0.54 mg/L. This method was used in the analysis of spiked arsenic species in tap and mineral water to demonstrate its usefulness. The results showed that both the recovery and the reproducibility of the developed method were acceptable.

Construction of hollow DNA/PLL microcapsule as a dual carrier for controlled delivery of DNA and drug

DNA/poly-L-lysine (PLL) capsules were constructed through a layer-by-layer (LbL) self-assembly of DNA and PLL on CaCO3 microparticles, and then used as dual carriers for DNA and drug after dissolution of carbonate cores. The permeability of DNA/PLL microcapsules was investigated with fluorescence probes with different molecular weights by confocal microscopy. The result revealed that the fluorescence probes were able to penetrate the capsule walls even its molecular weight up to 150 kDa. The resultant capsules were used to load drug model molecules-fluorescein isothiocyanate (FITC)-dextran (4 kDa) via spontaneous deposition mechanism.

C-60 trianion-mediated electrocatalysis and amperometric sensing of hydrogen peroxide

Heterogeneous electrocatalytic reduction of hydrogen peroxide (H2O2) by C-60 is reported for the first time. C-60 is embedded in tetra octyl ammonium bromide (TOAB) film and is characterized by scanning electron microscopy and cyclic voltammetry. Electrocatalytic studies show that the trianion of C-60 mediates the electrocatalytic reduction of H2O2 in aqueous solution containing 0.1 M KCl. Application of such film modified electrode as an amperometric sensor for H2O2 determination is also examined.

Microarray-based Raman spectroscopic assay for kinase inhibition by gold nanoparticle probes

In this paper, a microarray-based surface-enhanced Raman spectroscopic (SERS) assay for detection of kinase functionality and inhibition has been reported. Biotinylated anti-phosphoserinen antibodies mark the phosphorylation and inhibition events and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry, followed by silver deposition for SERS signal enhancement. The avidin conjugated fluorescein is used as SERS probe. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), its well known substrate, kemptide, and three inhibitors, H89, HA1077, and KN62 have been chosen here to establish the SERS assay. As expected, highly selective inhibition of PKA is demonstrated with the inhibitor H89 and the inhibition assay enable to detect kinase inhibition as well as derive IC50 (half maximal inhibitory concentration) plots.