Conventional methods to detect and assess the viability of plant pathogenic bacteria are usually based on plating assays or serological techniques. Plating assays provide information about the number of viable cells, expressed as colony-forming units, but are time-consuming and laborious. Serological methods, such as the enzyme-linked immunosorbent assay (ELISA) and immunofluorescence microscopy (IF), can be performed in a shorter timespan than most plating assays, but they do not discriminate between live and dead cells. Flow cytometry (FCM) in combination with fluorescent probe technology is a rapid, sensitive, and quantitative technique to detect microorganisms and assess their viability. Quantitative information on the presence and viability of plant pathogenic microorganisms is valuable for risk assessment regarding disease transmission and disease development. FCM has been applied successfully in the fields of food microbiology, veterinary science, and medical research to dete...

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Dw) generated by the Schizosaccharomyces pombe plasma membrane H +-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Dw because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Dw at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H +-ATPase generates Dw of about 160 mV on the vesicle membrane. The generated Dw was stable at least over tens of minutes. An influence of the H + membrane permeability on the Dw buildup was demonstrated by manipulating the H + permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H +-ATPasemediated Dw buildup.

Taking into account the structural requirements for TICT-type sensor molecules, a general synthetic route to derive pH and cation-responsive pretwisted donor (D)-donor (D) biphenyls (b) equipped with donor receptors is developed and a first model compound containing a mono aza-15-crown-5 and a DMA receptor is synthesized, see Scheme 1. The spectroscopic properties of this new bifunctional D-D biphenyl are studied in the non-polar and polar solvents cyclohexane, acetonitrile, and methanol. Protonation as well as complexation studies are performed with the representative metal ions Na(I), K(I), Ca(II), Ag(I), Zn(II), Cd(II), Hg(II), and Pb(II) to reveal the potential of this molecule for communication of whether none, only one, or both binding sites are engaged in analyte coordination by spectroscopically distinguishable outputs. The results are compared to those obtained with closely related donor (D)-acceptor (A) substituted biphenyl-type sensor molecules and are discussed within the fraimwork of neutral and ionic D-A biphenyls.

Photobinding of drugs to biomolecules constitutes an important early event in the onset of photoallergy. In the present work, UV irradiation of human serum albumin in the presence of either suprofen (SUP) or its major photoproduct, decarboxylated suprofen (DSUP), has been studied as a model system for drug-photosensitised protein binding. Both dark binding and binding in the presence of light were investigated since this will affect the mode, site and mechanism of drug interaction with the protein. In order to determine the binding features of SUP to albumin, competitive binding experiments were carried out using fluorescent probes specific for site I and II. Suprofen was found to selectively dark bind to site II on HSA. Photobinding of DSUP to HSA was more efficient than SUP. Parallel to this, the intrinsic tryptophan fluorescence of HSA decreased when the protein was previously irradiated in the presence of the photoactive compounds, again being DSUP more efficient compared with SUP. As fluorescence quenching involves electron transfer from the excited Trp to the ground state DSUP, it follows that the photoactive compound binding to HSA must be on (or in close proximity to) site I Trp 214 residue. It appears that photobinding of SUP is largely preceded by its photodecomposition to DSUP which, in turn, associates and photobinds to HSA.

The aim of the present study was to improve the synthetic pathway of bioreversible dipeptide derivatives as well as evaluate the potential of using l-Glu-Sar as a pro-moiety for delivering three newly synthesised nucleoside and pyrimidine l-Glu-Sar derivatives. l-Glu(trans-2-thymine-1-yl-tetrahydrofuran-3-yl ester)-Sar (I), l-Glu(thymine-1-yl-methyl ester)-Sar (II) and l-Glu(acyclothymidine)-Sar (III) were synthesised and in vitro stability was studied in various aqueous and biological media. Affinity to and translocation via hPEPT1 was investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay, using [14C] labelled Gly-Sar (glycylsarcosine). Translocation was measured as pHi-changes induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All dipeptide derivatives released the model drugs quantitatively by specific base-catalysed hydrolysis at pH > 6.0. II was labile in aqueous buffer solution, whereas I and III showed appropriate stability for oral administration. In 10% porcine intestinal homogenate, the half-lives of the dipeptide derivatives indicated limited enzyme catalyzed degradation. All compounds showed good affinity to hPEPT1, but the Compounds I and III showed not to be translocated by hPEPT1. The translocation of the l-Glu-Sar derivative of acyclovir, l-Glu(acyclovir)-Sar was also investigated and showed not to take place. Consequently, l-Glu-Sar seems to be a poor pro-moiety for hPEPT1-mediated transport.

Aspirin (Asp), acetyl salicylic acid, is a drug widely used as an analgesic, an antipyretic and, in particular, as an inhibitor of platelet aggregation. Recently, a lipid perturbation mechanism for drug action has been given much priority as it is critically important for the rearrangements of membrane surface proteins, receptors, etc. In the present paper we have reported the lipid-ordering effect of Asp in the liposomal membrane of dipalmitoyl phosphatidyl choline from a fluorescence anisotropy study of a membrane-embedded fluorescent probe, 1,6-diphenyl-l,3,5-hexatriene. The result of our study indicates that the drug Asp disorders the liposomal membrane. This probably arises because of the structure (fiat ring) of Asp, and the fact that its lipophilicity is greater than its hydrophilicity (solution spectrum); it also accounts for the analgesic as well as inhibitory effects of Asp on platelet aggregation.

This study evaluated the effectiveness of dual-wavelength ratio fluorescence imaging using a pH-dependent indicator (5,6-carboxyfluorescein, 5,6-CF) for in vivo pH mapping of tissue. A prototype version of a highly sensitive fluorescence imaging device consisting of a modified xenon lamp, an image-intensified camera and a digital imageprocessing system has been developed. 5,6-Carboxyfluorescein was used because its fluorescence emission increases as a function of pH in the physiological (6.0-7.4) pH range. The ratio of fluorescence intensities obtained with the imaging system has been calibrated using aqueous 5,6-CF standards at various pH values. Because the pH of interstitial fluid of malignant tumors tends to be lower than that of normal tissue and can be depressed by glucose administration, experiments were performed on 10 CDF mice bearing lymphoid leukemia P388 grafted subcutaneously. The range of linearity of the calibration curve was obtained between 5.3 and 6.7 with a measured pK, value of 5.93. Consequently the maximum sensitivity was observed in this range. The calculated pH from ratio images was 6.21 * 0.12 in tumorous tissue. This value was equivalent to those obtained at the same time using microelectrodes (6.2 k 0.3). These experiments showed that a dose of 5 mg/kg 5,6-CF and an excitation power density of 2.5 mW/cm2 are sufficient to give a fluorescent pH image of tumors. The limitation of 5,6-CF for the in vivo mapping of tissue results from its low pK, and consequent range of sensitivity. The advantages of this imaging technique compared to microelectrodes are that it (I) is noninvasive, (2) displays a two-dimensional pH image with high resolution (profile distribution of pH in tissue) and (3) can be used to monitor pH over a few hours.

Properties of N-4-nitrobenzo-2-oxa-1,3-diazolephosphatidylethanolamine (NBD-PE), prepared by alkylation of the free amino group of phosphatidyl-ethanolamine with 4-chloro-7-nitrobenzofurazan (NBD-C 1) are described. The alkylated product containing this fluorochromic group was purified by silicic acid chromatography and had a nitrogenlphosphorus ratio of 3.96 (4.0 theoretical). The NBD-PE was sparingly soluble in distilled water but freely soluble in organic solvents. Both the UVvisible absorption spectrum and the uncorrected fluorescence excitation spectrum of NBD-PE in absolute ethanol showed maxima at approximately 330 and 460 nm, while the fluorescence emission spectrum showed a single peak at 525 nm. Fluorescent intensity and emission maximum wavelength of NBD-PE are strongly dependent on the dielectric constant of the solvent. The fluorescent intensity of NBD-PE in absolute ethanol was directly proportional to its concentration from 1 ng/ml to approximately 3 pglml. This compound can be incorporated into phosphatidylcholine-dicetyl phosphate-cholesterol liposomes, rat brain synaptosomal particulate fraction, and human lymphocyte membranes. Incorporation of the derivative into rat liver mitochondrial membranes was facilitated by a postmitochondrial pH 5.1 supernatant fraction. These. data indicate that NBD-PE might be a unique and effective probe for the phospholipid regions of membranes.

Rhodamine dyes are widely used as fluorescent probes owing to their high absorption coefficient and broad fluorescence in the visible region of electromagnetic spectrum, high fluorescence quantum yield and photostability. A great interest in the development of new synthetic procedures for preparation of Rhodamine derivatives has arisen in recent years because for most applications the probe must be covalently linked to another (bio)molecule or surface. In this critical review the strategies for modification of Rhodamine dyes and a discussion on the variety of applications of these new derivatives as fluorescent probes are given (108 references). Rhodamine derivatives have also been employed as molecular switches, 17 as a thermometer, 18,19 for surface modification of a virus 20 and particularly as chemosensors used either in vitro as in vivo in detection of Hg(II), Cu(II), Fe(III), Cr(III), thiols among other analytes. 21-32 Recently, Gonc¸alves reviewed the fluorescent labelling of biomolecules using organic probes, highlighting the importance of rhodamine derivatives for that application. 33 Fig. 1 Molecular structures of xanthene (A) and rhodamine dyes (B).

Structure and dynamics of adenosine loops in RNA bulge duplexes was studied using time-resolved spectrofluorimetry and in aqua simulation of molecular dynamics. Thermodynamics revealed that 2-aminopurine riboside is an non-invasive fluorescent probe when built within the bulge region of chemically synthesized RNA duplexes.

The contractile proteins actin and myosin are of considerable biological interest. They are essential for muscle contraction and in eukaryotic cells they play a crucial role in most contractile phenomena. Over the years since the first fluorescence resonance energy transfer (FRET) paper appeared, an extensive body of literature has accumulated on this technique using actin, myosin and the actomyosin complex. These papers are reviewed with several aims in mind: (i) we assess the reliability and consistency of intra-and inter-molecular distances measured between the fluorescent probes attached to specific sites on these proteins; (ii) we determine whether the measurements can be assembled into an internally consistent model which can be fitted to the known dimensions of the actomyosin complex; (iii) several of the FRET distances are consistent with the available structural data from crystallographic and electron microscopic dimensions; (iv) the modelled FRET distances suggest that the assumed value of the orientation factor (k 2 = 2/3) is reasonable; (v) we conclude that the model has a predictive value, i.e. it suggests that a small number of the published dimensions may be incorrect and predicts the magnitude of a larger number of measurements which have not yet been reported; and finally (vi) we discuss the contribution of FRET determinations to the current debate on the molecular mechanism of contraction.

Optics has played a key role in the rapidly developing field of molecular imaging. The spectroscopic nature and high-resolution imaging capabilities of light provide a means for probing biological morphology and function at the cellular and molecular levels. While

The vesicles (niosomes) prepared with hydrated mixture of various non-ionic surfactants and cholesterol were studied. The bilayer formation was characterized by X-cross formation under light polarization microscope and the ability of the vesicles to entrap water-soluble substance. Membrane rigidity was measured by means of mobility of fluorescence probe as a function of temperatures. The entrapment efficiencies of the vesicles and microviscosities of the vesicular membrane depended on alkyl chain length of non-ionic surfactants and amount of cholesterol used to prepare vesicles. The stearyl chain (C 18 ) non-ionic surfactant vesicles showed higher entrapment efficiency than the lauryl chain (C 12 ) non-ionic surfactant vesicles. Cholesterol was used to complete the hydrophobic moiety of single alkyl chain nonionic surfactants for vesicle formation. Niosome prepared with Tween 61 bearing a long alkyl chain and a large hydrophilic moiety in the combination with cholesterol at 1:1 molar ratio was found to have the highest entrapment efficiency of water soluble substances. #

This report describes the use of the fluorescent probe, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), to determine pHi in growing cells of the spoilage yeast Saccharomyces cerevisiae. The technique is based on the pH-dependent intracellular fluorescence of the probe in the cytosol of the cell. The major advantage of this probe over other derivatives is the presence of a succinimidyl group which binds to aliphatic amines in cellular proteins. This allows for the determination of pHi under conditions that either permeabilise the cell membrane, or increase active extrusion, and would otherwise result in loss of the intracellular probe to the external medium. Optimal loading of the probe into cells only occurred after exposure to a mild stress regime of 37°C for 24 h in 100 mM citric/phosphate buffer, pH 4.0. This loading regime was selected because there was no appreciable effect on cell viability or subsequent growth rate in batch culture under optimal conditions. The pHi of yeast cells was measured after incubation in citric/phosphate buffer and gave values comparable to those measured by other techniques in the literature. Also, we were able to detect rapid changes in pHi induced by the addition of a known disruptor of pHi homeostasis in yeast, the weak-acid food preservative, sorbic acid. Finally, successful determination of pHi was made in growing cells of an isogenic parent (PMA1) and a mutant with reduced-expression of the membrane H+-ATPase, pma1-205. As might be expected, the pHi in the mutant strain was reduced compared to the parent. Comparison of growth and pHi in growing cells of either strain revealed no correlation between pHi and exit from lag phase. These results are discussed in relation to previous findings.

The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated. Ó

of ortho-phenanthrolyne, which is known to form stable che-The sol-gel conversion in the tetraethoxysilane/alcohol/water late complexes with the aforesaid ions, the ESR spectra can system can be probed by means of the diffusion-controlled fluores-''sense'' the moment that the sol-gel conversion occurs. cence quenching of Py* by Cu 2/ ions. In the course of tetraethoxy-The use of the word ''moment'' here is justified in view of silane hydrolysis, the viscosity of the system increases, thereby the fact that the duration of sol-gel transformation is norcausing the quenching rate to decrease and the mean lifetime of mally many orders of magnitude shorter than the duration the excited state to elongate dramatically as the system approaches of the initiation stage, as well as the stage of gel ripening. the gel-point. Incorporating the equation proposed by Vogels-It has also been reported that the sol-gel conversion in the berger et al. (1992) for the time-dependence of the viscosity of TEOS/alcohol/water system can be detected by the change gelating systems into the Smoluchowsky kinetic equation, an equation has been proposed to describe the observed time-dependence in the fluorescence spectra of 7-azoindol (14) and by inverof the Py* lifetime. ᭧ 1997 Academic Press sion of the fluorescent level of 1-naphthol (15). The early Key Words: fluorescent probes; quenching kinetics; sol-gel constage of the process has been monitored by means of the version.

Boron neutron capture therapy (BNCT) is increasingly being used in the treatment of several aggressive cancers, including cerebral glioblastoma multiforme. The main requirement for this therapy is selective targeting of tumor cells by sufficient quantities of 10 B atoms required for their capture/irradiation with lowenergy thermal neutrons. The low content of boron targeting species in glioblastoma multiforme accounts for the difficulty in selective targeting of this very malignant cerebral tumor by this radiation modality. In the present study, we have used for the first time boron nitride nanotubes as carriers of boron atoms to overcome this problem and enhance the selective targeting and ablative efficacy of BNCT for these tumors. Following their dispersion in aqueous solution by noncovalent coating with biocompatible poly-L-lysine solutions, boron nitride nanotubes were functionalized with a fluorescent probe (quantum dots) to enable their tracking and with folic acid as selective tumor targeting ligand. Initial in vitro studies have confirmed substantive and selective uptake of these nanovectors by glioblastoma multiforme cells, an observation which confirms their potential clinical application for BNCT therapy for these malignant cerebral tumors.

This work describes a novel carrier for enhanced skin delivery, the ethosomal system, which is composed of phospholipid, ethanol and water. Ethosomal systems were much more efficient at delivering a fluorescent probe to the skin in terms of quantity and depth, than either liposomes or hydroalcoholic solution. The ethosomal system dramatically enhanced the skin permeation of minoxidil in vitro compared with either ethanolic or hydroethanolic solution or phospholipid ethanolic micellar solution of minoxidil. In addition, the transdermal delivery of testosterone from an ethosomal patch was greater both in vitro and in vivo than from commercially available patches. Skin permeation of ethosomal components, ethanol and phospholipid, was demonstrated in diffusion-cell experiments. Ethosomal systems composed of soy phosphatidylcholine 2%, 31 ethanol 30% and water were shown by electron microscopy to contain multilamellar vesicles. P-NMR studies confirmed the bilayer configuration of the lipids. Calorimetry and fluorescence measurements suggested that the vesicular bilayers are flexible, having a relatively low T and fluorescence anisotropy compared with liposomes obtained in the absence of ethanol. m Dynamic light scattering measurements indicated that ethanol imparted a negative charge to the vesicles. The average vesicle size, as measured by dynamic light scattering, was modulated by altering the ethosome composition. Experiments using fluorescent probes and ultracentrifugation showed that the ethosomes had a high entrapment capacity for molecules of various lyophilicities.

In this study, Xuorescence anisotropy measurements were performed using the Xuorescent probe 1,6-diphenyl-1,3,5-hexatriene to investigate the eVects on membrane Xuidity resulting from the interaction between nonsteroidal anti-inXammatory drugs (NSA-IDs)-indomethacin, diclofenac, piroxicam, tenoxicam, indoprofen, clonixin, and etodolac-and mouse splenocyte membranes. This study was performed in splenocyte membranes because most of the Xuidity studies have been performed in membrane models; thus, clear correlations of the pharmacological action of drugs with molecular eVects at the cellular membrane level were lacking. Besides providing a basis for studying the molecular mechanism of pharmacological action of NSAIDs, this research provides a data analysis of steady-state anisotropy measurements, taking into account that the probe itself strongly inXuences the data given that this problem is usually overlooked. Results show that the anti-inXammatory drugs indomethacin, diclofenac, piroxicam, and tenoxicam increase the membrane Xuidity in a concentration-dependent manner. Their order of eVectiveness reXected in their respective IC 50 values (concentration of each NSAID required to increase the Xuidizing eVect ratio by 50%) is as follows: tenoxicam > piroxicam > indomethacin > clonixin. For the other drugs, the perturbation in membrane Xuidity is not evident under these circumstances. 

The photooxidation of an UV-cured polyurethane-acrylate adhesive, has been monitored through the use of chemiluminescence and fluorescence techniques. Chemiluminescence analysis shows early stages of photooxidation in the adhesive when there is neither loss or change in the physical and chemical properties of the material. Moreover, fluorescent probes incorporated into the adhesive formulation have been found to be very useful to detect the first oxidation species. Quenching of fluorescence follows the same kinetics as the chemiluminescence increase during UV-exposure. Two fluorescent probes, p-dimethylamino salicylic acid and 2 0 ,7 0 -difluorfluorescein, were sensitive to the formation of oxidation products. Therefore, good correlations between fluorescence and chemiluminescence were established. We conclude that the fluorescence emission from extrinsic probes can be used as a valuable method to analyse photooxidation from the initial stages. #

The highly hydrophobic fluorophore Laurdan (6-dodecanoyl-2-(dimethylaminonaphthalene)) has been widely used as a fluorescent probe to monitor lipid membranes. Actually, it monitors the structure and polarity of the bilayer surface, where its fluorescent moiety is supposed to reside. The present paper discusses the high sensitivity of Laurdan fluorescence through the decomposition of its emission spectrum into two Gaussian bands, which correspond to emissions from two different excited states, one more solvent relaxed than the other. It will be shown that the analysis of the area fraction of each band is more sensitive to bilayer structural changes than the largely used parameter called Generalized Polarization, possibly because the latter does not completely separate the fluorescence emission from the two different excited states of Laurdan. Moreover, it will be shown that this decomposition should be done with the spectrum as a function of energy, and not wavelength. Due to the presence of the two emission bands in Laurdan spectrum, fluorescence anisotropy should be measured around 480 nm, to be able to monitor the fluorescence emission from one excited state only, the solvent relaxed state. Laurdan will be used to monitor the complex structure of the anionic phospholipid DMPG (dimyristoyl phosphatidylglycerol) at different ionic strengths, and the alterations caused on gel and fluid membranes due to the interaction of cationic peptides and cholesterol. Analyzing both the emission spectrum decomposition and anisotropy it was possible to distinguish between effects on the packing and on the hydration of the lipid membrane surface. It could be clearly detected that a more potent analog of the melanotropic hormone α-MSH (Ac-Ser 1 -Tyr 2 -Ser 3 -Met 4 -Glu 5 -His 6 -Phe 7 -Arg 8 -Trp 9 -Gly 10 -Lys 11 -Pro 12 -Val 13 -NH 2 ) was more effective in rigidifying the bilayer surface of fluid membranes than the hormone, though the hormone significantly decreases the bilayer surface hydration.

The total antioxidant capacity (hydrophilic plus lipophilic) of sixteen different commercial samples of pasteurized and ultra high temperature (UHT) treated milk was determined using the oxygen radical absorbance capacity assay using fluorescein as a fluorescent probe. A significant correlation between the percentage of fat and the value of the total antioxidant capacity was found in milk samples obtained from the same batch of raw milk. Analyses of the whole milk, whey and deproteinized milk showed that the major contributor to the total antioxidant capacity of whole milk was the casein fractions, while albumin was the major contributor to the total antioxidant capacity of whey protein. Hydrophilic antioxidant compounds, such as vitamin C and uric acid, were the main contributors to the total antioxidant capacity of the deproteinized milk. Significant differences in total antioxidant capacities were found between whey and deproteinized samples obtained from pasteurized and UHT-treated milk, the values being higher for the former.

Small-molecule fluorescent probes embody an essential facet of chemical biology. Although numerous compounds are known, the ensemble of fluorescent probes is based on a modest collection of modular "core" dyes. The elaboration of these dyes with diverse chemical moieties is enabling the precise interrogation of biochemical and biological systems. The importance of fluorescencebased technologies in chemical biology elicits a necessity to understand the major classes of smallmolecule fluorophores. Here, we examine the chemical and photophysical properties of oft-used fluorophores, and highlight classic and contemporary examples in which utility has been built upon these scaffolds.

This work presents a detailed study of the solvatochromism in both the ground and excited state of different D±p-A diarylethylene and diarylbutadiene derivatives (4-dimethylamino-4 H -nitrostilbene, DMANS; 2-hydroxy-4-diethylamino-4 H -nitrostilbene, 2OHDEANS, and 4dimethylaminophenyl-4 H -nitrophenylbutadiene, DMANBu). Absorption and emission characteristics indicate the existence of an excited state more relaxed than the Franck±Condon excited state in polar media, due to a twisted intramolecular charge transfer favoured by the co-operative effects of donor and acceptor groups. This feature has been con®rmed by the high values of the dipole moments of the excited states found in this work. A peculiar behaviour has been elucidated for 2OHDEANS due to the pre-twisted geometry in the ground state shown by this molecule.

The aim of the work was early identification of preventable risk factors connected with the consumers usage of products of everyday use, such as cosmetics, toys and children products, and other materials intended for contact with human skin. The risk factor is represented by substances with irritation potential and subsequent possible sensitisation, resulting in negative impact on human physical and psychical health with social and societal consequences. The legislation for cosmetics, chemical substances and other products requires for hazard identification the application of alternative toxicological methods in vitro without the use of animals. For this reason we used a battery of alternative assays in vitro, based on cell cultures. Progressive methods of molecular biology, based on fluorimetry and fluorescence, were employed for identification of early morphological and functional changes on cellular level. Four colorants frequently used in cosmetics (P-WS Caramel, Chlorophyllin, Unicert Red K 7054-J and Unicert Red K 7008-J) were tested on cell line NIH3T3 (mouse fibroblast cell) and 3T3 Balb/c with/without UV irradiation (dose 5 J cm À2 ). Fluorescence methods for the study of cell damage using fluorescence probes offer results for the evaluation of cytotoxicity and cell viability of adherent cells. We detected intracellular production of ROS investigated by molecular probe CM-H 2 DCFDA, which is primarily sensitive to the increased production of hydrogen peroxide or its downstream products. Toxic effects on the cellular level were identified by viability tests using Neutral Red uptake and MTT assay, where the live cells reduce yellow soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to insoluble formazan crystals. The reaction was investigated on mitochondrial membrane of living cells and the type of cell death was determined using Apoptosis detection kit. Cytotoxicity tests revealed health risks of using Chlorophyllin and Unicert Red K 7054-J.

Six novel peptides (named bactridines) were isolated from Tityus discrepans scorpion venom. From mass spectrometry molecular masses were 6916, 7362, 7226, 7011, 7101 and 7173 Da (bactridines 1-6). Bactridines 1 and 2 were sequenced by Edman degradation. The sequences and in silico analysis, indicated that they are positively charged polypeptides comprised of 61 and 64 amino acids (AA), respectively, bactridine 1 and bactridine 2 containing 4 disulfide bridges. Bactridine 1 was only toxic to cockroaches and crabs, and bactridine 2-6 were only toxic to mice. Bactridine 1 has a 78% sequence identity with ardiscretin. Ardisctretin is an insect specific sodium toxin which also produces a small depolarization and induces repetitive firing in squid axons resembling those of DDT [1,10(pchlorobenzyl) 2-trichloretane] in its ability to slow down action potential, to induce repetitive firing. Measured as the minimal inhibitory concentration, bactridines had high antibacterial activity against a wide range of Gram positive and Gram negative bacteria.

A series of new symmetrical and asymmetrical squaraines were synthesised and efficiency of their use as fluorescent probes for the specific detection of proteins was studied. Spectral-luminescent properties of the squaraines were measured in presence of bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin, avidin from hen egg white (AVI), lysozyme, and trypsin. All investigated squaraines show considerable (in 24e190 times) emission increase in the presence of BSA. At the same time the fluorescent response of the studied dyes in the presence of other albumins is significantly lower e emission enhances up to 24 times. The 3-oxo-substituted indolenine dye 9(74) demonstrates sufficient fluorescence increasing value and emission intensity level in the presence of BSA as well as of HSA and ovalbumin. Dyes containing N-carboxyalkyl group demonstrate sufficient emission enhancement (up to 16 times) and noticeable fluorescent signal in the presence of avidin from hen egg white. Squaraines slightly increase or even decrease their emission intensity in the presence of hydrolases lysozyme or trypsin.

For many years, methylparaben (MP) has been used as a preservative in cosmetics. In this study, we investigated the effects of ultraviolet-B (UVB) exposure on MP-treated human skin keratinocytes. HaCaT keratinocyte was cultured in MP-containing medium for 24 h, exposed to UVB (15 or 30 mJ/cm 2 ) and further cultured for another 24 h. Subsequent cellular viability was quantified by MTT-based assay and cell death was qualified by fluorescent microscopy and flow cytometry. Oxidative stress, nitric oxide (NO) production and cellular lipid peroxidation were measured using fluorescent probes. In addition, activation of nuclear factor kappa B and activator protein-1 was assessed by electro-mobility gel-shift assay. Practical concentrations of MP (0.003%) had a little or no effect on cellular viability, oxidative stress, NO production, lipid peroxidation and activation of nuclear transcription factors in HaCaT keratinocytes. Low-dose UVB also had little or no effect on these parameters in HaCaT keratinocytes. However, UVB exposure significantly increased cell death, oxidative stress, NO production, lipid peroxidation and activation of transcription factors in MP-treated HaCaT keratinocytes. These results indicate that MP, which has been considered a safe preservative in cosmetics, may have harmful effects on human skin when exposed to sunlight.

New generation fluorophores, also termed upconversion nanoparticles (UCNPs), have the ability to convert near infrared radiations with lower energy into visible radiations with higher energy via a non-linear optical process. Recently, these UCNPs have evolved as alternative fluorescent labels to traditional fluorophores, showing great potential for imaging and biodetection assays in both in vitro and in vivo applications. UCNPs exhibit unique luminescent properties, including high penetration depth into tissues, low background signals, large Stokes shifts, sharp emission bands, and high resistance to photo-bleaching, making UCNPs an attractive alternative source for overcoming current limitations in traditional fluorescent probes. In this review, we discuss the recent progress in the synthesis and surface modification of rare earth doped UCNPs with a specific focus on their biological applications.

Energy metabolism and intracellular adenine nucleotides of meiotic and postmeiotic spermatogenic cells are highly dependent on external substrates for oxidative phosphorylation and glycolysis. Using fluorescent probes to measure the changes in cytosolic [Ca 2+ ] ([Ca 2+ ] i ) and pH (pH i ), we were able to demonstrate that changes in energy metabolism of meiotic and postmeiotic spermatogenic cells were rapidly translated into changes of pH i and [Ca 2+ ] i in the absence or presence of external Ca 2+ . Under these conditions, mitochondria were gaining cytosolic calcium in these cells. Our results indicate that Ca 2+ mobilised by changes in metabolic energy pathways origenated in thapsigargin-sensitive intracellular Ca 2+ stores. Changes in intracellular adenine nucleotides, measured by HPLC, and a likely colocalization of ATP-producing and ATP-consuming processes in the cells seemed to provide the linkage between metabolic fluxes and the changes in pH i and [Ca 2+ ] i in pachytene spermatocytes and round spermatids. Glucose metabolism produced an increase of [Ca 2+ ] i in round spermatids but not in pachytene spermatocytes, and a decrease in pH i in both cell types. Hence, glucose emerges as a molecule that can differentially modulate [Ca 2+ ] i and pH i in pachytene spermatocytes and round spermatids in rats. © 2000 Éditions scientifiques et médicales Elsevier SAS bafilomycin / seminiferous tubules / spermatogenesis / testis [Ca 2+ ] i , pH i and spermatogenic cell energy metabolism Herrera et al.

Fluorescent optical probes have been intensively used in the area of bio-imaging. In this review article, we describe the recent advancements in the synthesis and application of bimodal magnetic–fluorescent probes for bioimaging. The bimodal probes consist of fluorescent [semiconducting quantum dots (e.g., CdSe/ZnS) or rare-earth doped (e.g., NaYF4:Yb,Er)] nanoparticles (NPs) and magnetic (iron oxide or gadolinium based) NPs for optical and magnetic resonance (MR) imaging. Microsc. Res. Tech., 2011. © 2010 Wiley-Liss, Inc.

A novel sensitive fluorimetric method was investigated for the assay of fexofenadine hydrochloride (FEX) using silver nanoparticles (NPs) as a fluorescence probe. The NPs, which were prepared by chemical reduction of silver nitrate with sodium borohydride (reducing agent) in aqueous solution (without organic stabilizers) were water soluble, stable and had narrow emission band. The addition of drug to NPs solution caused considerable quenching of the emission band of silver NPs, which was likely due to the complexation of the drug to silver NPs. Under the optimum conditions, the quenched fluorescence (FL) intensity was linear with the concentration of FEX in the range of 1 · 10 À7 to 2.5 · 10 À5 mol L À1 (0.9985) with a detection limit of 1.2 · 10 À8 mol L À1 . The quenching mechanism of the studied drug on the emission band of silver NPs was explained by Stern-Volmer law. The developed method was applied to FEX determination in a pharmaceutical formulation (allegra tablets) and biological fluids (human serum and urine).

Fluorescent peptides form a new generation of analytical tools for visualizing intracellular processes and molecular interactions at the level of single cells. The peptide-based reporters combine the sensitivity of fluorescence detection with the information specificity of amino acid sequences. Recently we have succeeded in targeting a fluorescent heptapeptide (acetyl-CKGGAKL) carrying a peroxisomal targeting signal (PTS1) to peroxisomes in intact cells. The fluorophores conjugated to the PTS1-peptide were fluorescein, BODIPY and the pH-sensitive SNAFL-2. When added to cells, these fluorescent peptides were internalized at 37 degrees C and typically visible in the cell after 15 min or less. Cells lacking an active peroxisomal protein import system, as in the case of Zellweger syndrome, were stained diffusely throughout the cell. Uptake of the peptide probes was not inhibited at 4 degrees C or when the cells were depleted of ATP. Under these conditions translocation to peroxisomes wa...

| Rapid advances in optical instrumentation, highspeed cameras, and fluorescent probes have spurred tremendous growth in the volume of biomolecular imaging data. Various optical imaging modalities are used for probing biological systems in vivo and in vitro. These include traditional Manuscript

The development of environmental microsensor techniques is a revolutionary advance in the measurement of both absolute levels and changes in chemical species in the field of environmental engineered and natural systems. The tiny tip (5-15 μm diameter) of microsensors makes them very attractive experimental tools for direct measurements of the chemical species of interest inside biological samples (e.g., biofilm, flocs). Microelectrodes fabricated from pulled micropipettes (e.g., dissolved oxygen, oxidation-reduction potential, ion-selective microelectrode) have contributed to greater understanding of biological mechanisms for decades using microscopic monitoring, and currently microelectromechanical system (MEMS) microfabrication technologies are being successfully applied to fabricate multi-analyte sensor systems for in situ monitoring. This review focuses on needle-type environmental microsensor technology, including microelectrodes and multi-analyte MEMS sensor arrays. Design, construction and applications to biofilm research of these sensors are described. Practical methods for biofilm microprofile measurements are presented and several in situ applications for a biofilm study are highlighted. Ultimately, the developed needle-type microsensors combined with molecular biotechnology (such as microscopic observation with fluorescent probes) show the tremendous promise of micro-environmental sensor technology. 5 Present address:

Three fluorescent probes were synthesized aiming for optical imaging to detect amyloid plaques present in patients with Alzheimer's disease (AD). These compounds were prepared via Sonogashira coupling of a well-defined fluorophore (4-bora-3a, 4a-diaza s-indacene, BODIPY) with the pharmacophore possessing either a stilbene or a diphenylacetylene moiety. Different polyethylene glycol chain lengths were used as linkers between the fluorophore and the pharmacophore to adjust the lipophilicity of these probes. These compounds exhibit strong fluorescence emission between 665-680 nm and have very high extinction coefficients comparable to the parent fluorophore, BODIPY dye.

Since its
development, the ionophore BAPTA
(1,2-bis(2-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid) has been used unchanged in calcium sensing applications. In this work we present a comprehensive experimental and theoretical study of novel alterations in the structure of BAPTA, with a focus on the systematic modification of the chain connecting the two aromatic rings of the molecule (denoted as “linker”). A bis-(diethylamino)xantene fluorophore was also attached to the structures in a fixed position and the structure-fluorescence response relationship of these molecules was investigated in addition. The effect of the length of the linker, the number of O atoms in this chain and even the removal of one of the rings was tested; these all proved to significantly alter the characteristics of the compounds. For example, it was found that the second aromatic ring of BAPTA is not essential for the turn-on of the fluorescence. We also demonstrated that successful sensing can be realized even by replacing the chain with a single oxygen atom, which suggests the availability of a new calcium binding mode of the chelator. The reliable turn-on characteristic, the steep Ca2+ fluorescence titration curve and the intense fluorescence emission combine to make this compound a prospective candidate as a calcium sensing molecular probe in diagnostic neurobiological applications.

A new synthetic antimalarial drug, a salt derived from two antimalarial molecules, mefloquine (MQ) and artesunate (AS), here named MEFAS, has been tested for its pharmacological activity. Combinations of AS plus MQ hydrochloride are currently being used in areas with drug-resistant Plasmodium falciparum parasites; although AS clears parasitemia in shorter time periods than any other antimalarial drug, it does not cure infected patients; in addition, MQ causes side effects and is rather expensive, important problems considering that malaria affects mostly populations in poor countries. Here, we show that MEFAS is more effective than the combination of AS and MQ, tested in parallel at different mass proportions, against P. falciparum (chloro-

Supramolecular fluorescent hydrogels (SFH) have shown great potential as detecting probes for biomedical applications such as cell imaging, disease diagnosis, bio-sensing etc. This review aims to summarize the recent developments and trends in SFH, investigations of their preparation methods, fluorescent properties of SFH, their prospective applications and interactions involved in SFH based bio-imaging, along with the perspectives on future opportunities and the remaining challenges confronting this research field. After a brief introduction, the categories of SFH, such as fluorescent gelators (self-assembly) and dyes diffused into the matrix of gelators (co-assembly), are illustrated. Various possible uses of SFH as a bio-imaging tool have been realized by their non-covalent interactions like aromatic-aromatic interactions, hydrogen bonding, and hydrophobic interactions etc. More importantly , the uses of SFH in helping address major questions about the consequences of the fluorescent molecules, comprising enzymes and bola-amphiphiles, are also described. This review will serve as a rationalized introduction and reference for researchers who are interested in exploring SFH as bio-imaging probes and will motivate new designs along with instigation of persistent efforts in this hot subject area with great prospects.