Fluorometric Data Research Articles

A Zinc(II) Schiff Base Complex as Fluorescent Chemosensor for the Selective and Sensitive Detection of Copper(II) in Aqueous Solution.

The development of chemosensors able to detect analytes in a variety of sample matrices through a low-cost, fast, and direct approach is of current interest in food, health, industrial, and environmental fields. This contribution presents a simple approach for the selective and sensitive detection of Cu2+ ions in aqueous solution based on a transmetalation process of a fluorescent substituted Zn(salmal) complex. Transmetalation is accompanied by relevant optical absorption changes and quenching of the fluorescence emission, leading to high selectivity and sensitivity of the chemosensor, with the advantage of not requiring any sample pretreatment or pH adjustment. Competitive experiments demonstrate a high selectivity of the chemosensor towards Cu2+ with respect to the most common metal cations as potential interferents. A limit of detection down to 0.20 μM and a dynamic linear range up to 40 μM are achieved from fluorometric data. By exploiting the fluorescence quenching upon formation of the copper(II) complex, simple paper-based sensor strips, visible to naked eyes under UV light, are used for the rapid, qualitative, and quantitative in situ detection of Cu2+ ions in aqueous solution over a wide concentration range, up to 10.0 mM, in specific environments, such as in industrial wastewater, where higher concentrations of Cu2+ ions can occur.

A Multi-Year Assessment of Phytoplankton Fluorescence in a Large Temperate River Reveals the Importance of Scale-Dependent Temporal Patterns Associated With Temperature and Other Physicochemical Variables

An integrated temporal study of a long-term ecological research and monitoring database of the St. Lawrence River was carried out. A long and mostly uninterrupted high temporal resolution record of fluorometric data from 2014 to 2018 was used to examine phytoplankton fluorometric variables at several scales and to identify temporal patterns and their main environmental drivers. Sets of temporal eigenvectors were used as modulating variables in a multiscale codependence analysis to relate the fluorometric variables and various environmental variables at different temporal scales. Fluorometric patterns of phytoplankton biomass in the St. Lawrence River are characterized by large, yearly-scale patterns driven by seasonal changes in water temperature, and to a lesser extent water discharge, over which finer-scale temporal patterns related to colored dissolved organic matter and weather variables can be discerned at shorter time scales. The results suggest that such an approach to characterize phytoplankton biomass in large rivers may be useful for processing large data sets from remote sensing efforts for detecting subtle large-scale changes in water quality due to land use practices and climate change.

Integration of In Situ and Remote Sensing Measurements for the Management of Harmful Cyanobacteria Blooms. A Lesson from a Strategic Multiple-Uses Reservoir (Lake Occhito, South Italy)

Harmful cyanobacteria blooms (HCBs) are one of the main water quality threats affecting reservoirs. Guidelines suggest integrating laboratory, real-time in situ, and remote sensing data in the monitoring of HCBs. However, this approach is still little adopted in institutional measuring programs. We demonstrated that this integration improves frequency and spatial resolution of the data collection. Data were from an intense HCB (Planktothrix rubescens), which occurred in a south Italy multiple-uses reservoir (Lake Occhito) between 2008 and 2009 and regarded both the lake and the irrigation network. Laboratory and in situ fluorometric data were related to satellite imagery, using simple linear regression models, to produce surface lake-wide maps reporting the distribution of both P. rubescens and microcystins. In the first node of the distribution network, microcystin concentrations (4–10 µg L−1) reached values potentially able to damage the culture and to accumulate during cultivation. Nevertheless, our study shows a decrease in the microcystin content with the distance from the lake (0.05 µg L−1 km−1), with a reduction of about 80% of the microcystin concentrations at the furthest tanks. Recent improvements in the spatial resolution (i.e., tens of meters) of satellite imagery allow us to monitor the main tanks of large and complex irrigation systems.

Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato.

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

Synthesis, biological evaluation and molecular docking studies on the DNA and BSA binding interactions of palladium(II) and platinum(II) complexes featuring amides of tetrazol-1-yl- and tetrazol-5-ylacetic acids

Novel palladium(II) and platinum(II) complexes (trans-[PdCl2L2] and trans-[PtCl2L2] {L = 2-(5-methyl-1H-tetrazol-1-yl)acetamide, N-(sec-butyl)-2-(5-methyl-1H-tetrazol-1-yl)acetamide, N-cyclopropyl-2-(5-methyl-1H-tetrazol-1-yl)acetamide, N-cyclohexyl-2-(5-methyl-1H-tetrazol-1-yl)acetamide, 2-(2-(tert-butyl)-2H-tetrazol-5-yl)acetamide and 2-(2-(tert-butyl)-2H-tetrazol-5-yl)-N-cyclohexylacetamide} 1b–6b and 1c–4c) were synthesized and characterized by elemental analyses (CHN), HRESI+-MS, 1H, 13C{1H}, 195Pt NMR and IR spectroscopies, DSC/TG analysis, as well as by X-ray single crystal diffraction {for (2-(5-methyl-1H-tetrazol-1-yl)acetamide) 1a·H2O, trans-[PdCl2(2-(5-methyl-1H-tetrazol-1-yl)acetamide)2] 1b, trans-[PdCl2(N-cyclopropyl-2-(5-methyl-1H-tetrazol-1-yl)acetamide)2] 3b and trans-[PdCl2(N-cyclohexyl-2-(5-methyl-1H-tetrazol-1-yl)acetamide)2] 4b}. The binding of complexes 2b and 5b to calf-thymus DNA (CT DNA) and BSA (bovine serum albumin) was studied by means of CD, UV and fluorometric techniques. According to the spectroscopic data, an interaction of the metal complexes with CT DNA was confirmed. A significant increase in the melting point of CT DNA in the presence of the metal complexes {ΔTm = 10.0 °C (for 2b), 12.5 °C (for 5b)} indicates a strong stabilization of the DNA helix. According to the fluorometric data, complex 2b exhibits an interaction with BSA. Additionally, molecular docking results suggest that complex 1b and its fully hydrolyzed form (trans-[Pd(H2O)2(2-(5-methyl-1H-tetrazol-1-yl)acetamide)2]) cooperatively interact with DNA, presumably via minor groove binding. The hydrolyzed form possesses a greater affinity to DNA. Also, there is an effective interaction of complex 1b and the corresponding hydrolyzed form to BSA via the two binding sites Trp134 and Trp213. Two complexes {trans-[PdCl2(N-(sec-butyl)-2-(5-methyl-1H-tetrazol-1-yl)acetamide)2] 2b and trans-[PdCl2(2-(2-(tert-butyl)-2H-tetrazol-5-yl)acetamide)2] 5b} demonstrate antiproliferative activity against human cancer cell lines, with IC50 = 45 ± 0.5 µM for 2b on HeLa and IC50 = 44.3 ± 0.8 µM for 5b on MCF-7. The complexes 4b, 4c and 6b were evaluated for their antimicrobial activity against Escherichia coli (strain КA796) and they showed significant activities, being greater than that of cisplatin. Complex 4b was the most effective, with the lowest MLC value of 0.25 µM and LC50 value of 0.075 µM.

Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations.

The glycosyltransferase HisDapGalNAcT2 is the key protein of the Escherichia coli (E. coli) SHuffle® T7 cell factory which was genetically engineered to allow glycosylation of a protein substrate in vivo. The specific activity of the glycosyltransferase requires time-intensive analytics, but is a critical process parameter. Therefore, it has to be monitored closely. This study evaluates fluorometric in situ monitoring as option to access this critical process parameter during complex E. coli fermentations. Partial least square regression (PLS) models were built based on the fluorometric data recorded during the EnPresso® B fermentations. Capable models for the prediction of glucose and acetate concentrations were built for these fermentations with rout mean squared errors for prediction (RMSEP) of 0.19 g·L−1 and 0.08 g·L−1, as well as for the prediction of the optical density (RMSEP 0.24). In situ monitoring of soluble enzyme to cell dry weight ratios (RMSEP 5.5 × 10−4 µg w/w) and specific activity of the glycosyltransferase (RMSEP 33.5 pmol·min−1·µg−1) proved to be challenging, since HisDapGalNAcT2 had to be extracted from the cells and purified. However, fluorescence spectroscopy, in combination with PLS modeling, proved to be feasible for in situ monitoring of complex expression systems.

Caracterización de las aguas del Mar Caribe occidental mediante clorofila por fluorescencia <i>in vivo</i>

This paper presents the characteristics of fluorescence for the Caribbean Sea. Hydrographic and fluorometric data were gathered at the upper mixed layer during an oceanographic campaign carried out in the summer of 2001, from the Yucatan Peninsula to Colombian waters. In vivo fluorescence profiles show a conspicuous subsurface chlorophyll maximum located at a depth of around 107 m. These profiles are characteristic of oligotrophic waters such as those of the western Caribbean Sea.

In situ Ca2+ titration in the fluorometric study of intracellular Ca2+ binding

Imaging with Ca2+-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca2+ signaling. The spatiotemporal evolution of intracellular free Ca2+ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca2+ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca2+ imaging data, and can be exploited to investigate endogenous Ca2+ buffer properties. Here we extended the use of Ca2+ fluorometry in the characterization of Ca2+ binding molecules within cells, building on a method of titration of intracellular Ca2+ binding sites in situ with measured amounts of Ca2+ entering through voltage-gated Ca2+ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca2+ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca2+ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca2+ buffer and dye, this method yielded estimates of the endogenous buffer concentration and Kd, the dye Kd, and the fraction of Ca2+ inaccessible cellular volume. The in situ Kd of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca2+-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca2+ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca2+ binding species. This method offers a versatile approach to the study of endogenous Ca2+ binding molecules in their physiological milieu.

Chlorophyll a spatial inference using artificial neural network from multispectral images and in situ measurements

Considering the importance of monitoring the water quality parameters, remote sensing is a practicable alternative to limnological variables detection, which interacts with electromagnetic radiation, called optically active components (OAC). Among these, the phytoplankton pigment chlorophyll a is the most representative pigment of photosynthetic activity in all classes of algae. In this sense, this work aims to develop a method of spatial inference of chlorophyll a concentration using Artificial Neural Networks (ANN). To achieve this purpose, a multispectral image and fluorometric measurements were used as input data. The multispectral image was processed and the net training and validation dataset were carefully chosen. From this, the neural net architecture and its parameters were defined to model the variable of interest. In the end of training phase, the trained network was applied to the image and a qualitative analysis was done. Thus, it was noticed that the integration of fluorometric and multispectral data provided good results in the chlorophyll a inference, when combined in a structure of artificial neural networks.

NADPH oxidase 2 activity modulates antigen processing and epitope repertoires (P5186)

Abstract Phagocytes such as macrophages and dendritic cells are essential to both the innate and acquired immune systems of mammals. To produce effective immune responses, material antigens must first be processed and presented by phagocytes. The importance of antigen processing is often overlooked, which is exemplified by current therapies focusing on downstream immune processes in T cells and B cells. However, these therapies often result in modulation of the immune system as a whole. Instead, it would be ideal to modulate antigen processing to prevent the generation of epitopes able to stimulate and re-stimulate specific T cells. Antigen processing within phagosomes is a dynamic process influenced by several environmental factors such as pH and oxidative burst. Of particular importance to our lab is the oxidative burst generated by the protein complex NADPH oxidase 2. Our lab has previously published data showing that regulation of NADPH oxidase 2 activity results in modulation of phagosomal proteolysis through modulation of cysteine cathepsin activity. We further hypothesize that protein antigen processing and epitope repertoire generation can be predicted and more importantly influenced by changing the activity of NADPH oxidase 2. Using a combination of fluorometric data, HPLC, and mass spectrometry we have begun to show, through preliminary data, that epitope repertoires can indeed be influenced without knocking down antigen processing and presentation as a whole.

Structure–activity relationships for the binding of polymyxins with human α-1-acid glycoprotein

Here, for the first time, we have characterized binding properties of the polymyxin class of antibiotics for human α-1-acid glycoprotein (AGP) using a combination of biophysical techniques. The binding affinity of colistin, polymyxin B, polymyxin B3, colistin methansulfonate, and colistin nona-peptide was determined by isothermal titration calorimetry (ITC), surface plasma resonance (SPR) and fluorometric assay methods. All assay techniques indicated colistin, polymyxin B and polymyxin B3 display a moderate binding affinity for AGP. ITC and SPR showed there was no detectable binding affinity for colistin methansulfonate and colistin nona-peptide, suggesting both the positive charges of the diaminobutyric acid (Dab) side chains and the N-terminal fatty acyl chain of the polymyxin molecule are required to drive binding to AGP. In addition, the ITC and fluorometric data suggested that endogenous lipidic substances bound to AGP provide part of the polymyxin binding surface. A molecular model of the polymyxin B3–AGP F1*S complex was presented that illustrates the pivotal role of the N-terminal fatty acyl chain and the D-Phe6-L-Leu7 hydrophobic motif of polymyxin B3 for binding to the cleft-like ligand binding cavity of AGP F1*S variant. The model conforms with the entropy driven binding interaction characterized by ITC which suggests hydrophobic interactions coupled to desolvation events and conformational changes are the primary driving force for polymyxins binding to AGP. Collectively, the data are consistent with a role of this acute-phase reactant protein in the transport of polymyxins in plasma.

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Remarkable commonalities of electrogenic and electroneutral Na+–phosphate cotransporters

Remarkable commonalities of electrogenic and electroneutral Na⁺–phosphate cotransporters

In Vitro Studies Demonstrate Anticancer Activity of an Alkaloid of the Plant Gelsemium sempervirens

The chemical structure of the main fluorescenting compound in the ethanolic extract (mother tincture) of the American yellow jasmine, Gelsemium sempervirens, was determined by employing (1)H nuclear magnetic resonance (NMR), (13)C NMR, mass spectroscopy, high-performance liquid chromatography (HPLC), correlation spectroscopy (COSY), and Fourier transform infrared (FTIR) spectroscopy analyses. Spectrofluorometric analysis has been made of the mother tincture and its agitated serial dilutions (up to 12th potency) prepared according to a homeopathic procedure in which serial, agitated dilutions were made separately in glass and polypropylene containers. The succussions were made by employing three different modes: hand jerk, sonication, and vortexing. The chemical formula of scopoletin, the main fluorescent compound, was determined to be C(10)H(8)O(4) having a molecular weight of 192.17. Significant differences were noted between the remedies prepared in the two types of containers. Further, a comparison between any two methods of agitation revealed significant differences in fluorometric data of remedies at certain potency levels. The biological (anticancer) action of the crude extract, the alkaloid scopoletin, and 2C potency of Gelsemium sp were tested in vitro on the HeLa cell line through fluorescence microscopy, the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and fluorescent activated cell sorting (FACS). The role of nanoparticles presumably derived from the containers, their orientation, and their interaction with the starting substance during the dynamization process initiated by different modes of agitation could possibly be attributed to the differences noted in the fluorometric data of potencies prepared in the two types of containers and among the three different means of succussion tested.

Post-Transcriptional Gene Silencing in the Root System of the Actinorhizal Tree Allocasuarina verticillata

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.

THE CHALLENGES OF IMPLEMENTING DISPERSANT EFFECTIVENESS MONITORING FOR A GLOBAL RESPONSE

ABSTRACT Recent developments in dispersant effectiveness monitoring have led to huge benefits for the response communities. Successfully interpreting dispersant effectiveness monitoring fluorometric data in situ has managed to speed scientific results to the Incident Command in almost real time which is becoming increasingly important in the promotion of dispersant as a response tool across the globe, particularly as more and more countries look to developing legislation permitting their use. The ability to present data linking dispersed oil to chemical dispersant is a compelling method of persuasion for their utilisation. OSRL/EARL is unique in that it is the only industry owned oil spill response company with a global remit, this presents a number of challenges in respect of the practicalities of responding with dispersant monitoring capabilities. The equipment surrounding the use of the fluorometer requires flexibility and portability dependant upon the circumstance of the spills that arise. With the possibility of application of dispersant operations resting upon the ability to monitor as soon as possible after the operations commence, the challenges surrounding the logistics of operation must be overcome, If dispersant monitoring equipment is delayed in its deployment it is not reasonable to delay application given the limited window of opportunity however aerial observations alone present a margin for human error. Monitoring dispersant effectiveness with the aid of fluorometry is still only a quantitative tool, although its capabilities are well respected, there is scope for further improvement to reduce its limitations. Currently the Turner 10 AU is the predominant tool in dispersant monitoring response; its replacement with a smaller in-situ fluorometer could not only improve the means of collating the data but also reduce the equipment that is required to be transported with the monitoring team. There are presently few obstacles that would prevent new fluorometer equipment being configured with the current software set up. This paper will look at how an international response organisation has prepared for dispersant monitoring and the tools it has put in place for operational deployment. The system is operated by response personnel rather than scientists so provides a practical solution to the problem based on their experience.

8-Hydroxyquinoline Derivatives as Fluorescent Sensors for Magnesium in Living Cells

Despite the key role of magnesium in many fundamental biological processes, knowledge about its intracellular regulation is still scarce, due to the lack of appropriate detection methods. Here, we report the spectroscopic and photochemical characterization of two diaza-18-crown-6 hydroxyquinoline derivatives (DCHQ) and we propose their application in total Mg(2+) assessment and in confocal imaging as effective Mg(2+) indicators. DCHQ derivatives 1 and 2 bind Mg(2+) with much higher affinity than other available probes (K(d) = 44 and 73 microM, respectively) and show a strong fluorescence increase upon binding. Remarkably, fluorescence output is not significantly affected by other divalent cations, most importantly Ca(2+), or by pH changes within the physiological range. Evidence is provided on the use of fluorometric data to derive total cellular Mg(2+) content, which is consistent with atomic absorption data. Furthermore, we show that DCHQ compounds can be effectively employed to map intracellular ion distribution and movements in live cells by confocal microscopy. A clear staining pattern consistent with known affinities of Mg(2+) for biological ligands is shown; moreover, changes in the fluorescence signal could be tracked following stimuli known to modify intracellular Mg(2+) concentration. These findings suggest that DCHQ derivatives may serve as new tools for the study of Mg(2+) regulation, allowing sensitive and straightforward detection of both static and dynamic signals.

Hydrophobically Modified Amphiphilic Block Copolymer Micelles in Non-Aqueous Polar Solvents. Fluorometric, Light Scattering and Computer-Based Monte Carlo Study

The micellization behavior of a hydrophobically modified polystyrene-block-poly(methacrylic acid) diblock copolymer, PS-N-PMA-A, tagged with naphthalene between blocks and with anthracene at the end of the PMA block, was studied in 1,4-dioxane-methanol mixtures by light scattering and fluorescence techniques. The behavior of a single-tagged sample, PS-N-PMA, and low-molar-mass analogues was studied for comparison. Methanol-rich mixtures with 1,4-dioxane are strong selective precipitants for PS. Multimolecular micelles with compact PS cores and PMA shells may be prepared indirectly by dialysis from 1,4-dioxane-rich mixtures, or by a slow titration of copolymer solutions in 1,4-dioxane-rich solvents with methanol under vigorous stirring. In tagged micelles, the naphthalene tag is trapped in nonpolar and fairly viscous core/shell interfacial region. In hydrophobically modified PS-N-PMA-A micelles, the hydrophobic anthracene at the ends of PMA blocks tends to avoid the bulk polar solvent and buries in the shell. The distribution of anthracene tags in the shell is a result of the enthalpy-to-entropy interplay. The measurements of direct nonradiative excitation energy transfer were performed to estimate the distribution of anthracene-tagged PMA ends in the shell. The experimental fluorometric data show that anthracene tags penetrate into the inner shell in methanol-rich solvents. Monte Carlo simulations were performed on model systems to get reference data for analysis of time-resolved fluorescence decay curves. A comparison of experimental and simulated decays indicates that hydrophobic traps return significantly deep into the shell (although not as deep as in aqueous media). The combined light scattering, fluorometric and computer simulation study shows that the conformational behavior of shell-forming PMA blocks in non-aqueous media is less affected by the presence of nonpolar traps than that in aqueous media.

Polystyrene/Poly(2-vinylpyridine) Heteroarm Star Copolymer Micelles in Aqueous Media and Onion Type Micelles Stabilized by Diblock Copolymers,

Micellization of polystyrene/poly(2-vinylpyridine) heteroarm star copolymers, PSnPVPn, in aqueous media has been studied by a combination of light scattering and fluorescence techniques. Three copolymer samples differing in numbers of arms and their length form micelles with compact hydrophobic cores and polyelectrolyte poly(2-vinylpyridine) shells. The association number decreases with increasing number of water-soluble arms and their length. Micellization equilibrium is kinetically frozen in aqueous media. The micellar cores are frozen and the behavior of the micellar solutions is controlled by the polyelectrolyte behavior of the polyelectrolyte shell. In the case of the present heteroarm stars with ca. 101 arms, the soluble and insoluble arms are strongly segregated and the micellar cores are formed of pure polystyrene as with micelles formed by linear diblock copolymers. As concerns the sample with several tens of arms, a significant steric hindrance, together with a low association number (ca. 4), does not allow for a complete segregation of polystyrene and the poly(2-vinylpyridine) arms. The micellar cores formed by the very hairy star copolymer with several tens of arms contain a fraction of poly(2-vinylpyridine) arms as shown by fluorescence measurements with solubilized pyrene. Alkalimetric titration of an acidic mixture of PSnPVPn micelles with a linear poly(2-vinylpyridine)-block-poly(ethylene oxide) copolymer, PVP−PEO, yields fairly monodisperse spherical onion-skin micelles. The onion-skin micelle has a compact spherical PS core deriving from the parent PSnPVPn micelle, a fairly thin and compact middle layer formed by PVP both from the parent micelle and PVP−PEO copolymer, and a protective PEO shell. The formation of onion-skin micelles is fully reversible and occurs suddenly and very rapidly at pH values higher than 4.8. The “onions” dissociate immediately into PSnPVPn micelles and PVP−PEO copolymer below pH 4.8. Evaluation of the alkalimetric titration data shows that only ca. 40% of the PVP units in the micellar shells are protonized (even in strongly acidic solutions that contain a large surplus of a strong acid, e.g., in 0.1 M HCl). This observation is in agreement with indirect fluorometric data which indicate that in the case of a weak polyelectrolyte, such as poly(2-vinylpyridine), the inner layer of the polyelectrolyte shell close to the core is not ionized.

Recombinant green fluorescent protein-expressing human cytomegalovirus as a tool for screening antiviral agents.

A recombinant human cytomegalovirus (AD169-GFP) expressing green fluorescent protein was generated by homologous recombination. Infection of human fibroblast cultures with AD169-GFP virus produced stable and readily detectable amounts of GFP signals which were quantitated by automated fluorometry. Hereby, high levels of sensitivity and reproducibility could be achieved, compared to those with the conventional plaque reduction assay. Antiviral activities were determined for four reference compounds as well as a set of putative novel cytomegalovirus inhibitors. The results obtained were exactly in line with the known characteristics of reference compounds and furthermore revealed distinct antiviral activities of novel in vitro inhibitors. The fluorometric data could be confirmed by GFP-based flow cytometry and fluorescence microscopy. In addition, laboratory virus variants derived from the recombinant AD169-GFP virus provided further possibilities for study of the characteristics of drug resistance. The GFP-based antiviral assay appeared to be very reliable for measuring virus-inhibitory effects in concentration- and time-dependent fashions and might also be adaptable for high-throughput screenings of cytomegalovirus-specific antiviral agents.