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PubMed1999

Determination of nitrite in waters by microplate fluorescence spectroscopy and HPLC with fluorescence detection.

A selective and versatile fluorescence spectroscopic method for the determination of nitrite in waters has been developed. Nitrite reacts in the presence of mineral acids with the nonfluorescent N-methyl-4-hydrazino-7-nitrobenzofurazan forming N-methyl-4-amino-7-nitrobenzofurazan, which can be detected by fluorescence spectroscopy with an excitation maximum at lambda = 468 nm and an emission maximum at lambda = 537 nm in acetonitrile. Three new methods based on this reaction have been developed: Direct fluorescence spectroscopy, HPLC/fluorescence, or HPLC with UV/vis detector may be selected as detection techniques. On microplates, high-throughput fluorescence spectroscopy is achieved, while HPLC/fluorescence provides lower limits of detection, and HPLC with UV/vis detection enables evaluation of the reaction with standard instrumentation. Different water samples were investigated using all detection modes, and a photometric standard procedure was successfully employed to validate the new methods with an independent technique.

Chromatography, High Pressure Liquid↗
PubMed1982

Fluorescently labeled myosin subfragment 1: identification of the kinetic step associated with the adenosine 5'-triphosphate induced fluorescence decrease.

Marsh, and Lowey [Marsh, D. J., & Lowey, S. (1980) Biochemistry 19, 774-784] introduced a fluorescence label, 5-(iodoacetamido)fluorescein (IAF), into the alkali 1 light chain of myosin subfragment 1 (S-1) without perturbing the K+-Ca2+, or actin-activated ATPase activities of the labeled S-1. They also found that the addition of MgATP to the labeled S-1 led to a 6-10% decrease in the fluorescence of the bound IAF label. In the present study, we investigated the pre-steady-state kinetics of the fluorescence decrease of the IAF label caused by the binding of MgATP to the labeled S-1. Our results show that most of the decrease in IAF fluorescence is caused by the ATP-hydrolysis step. This implies that a conformational change in the S-1 molecular associated with ATP hydrolysis has led to a change in the microenvironment of the IAF fluorophore bound to the alkali 1 light chain.

Actins↗
PubMed2002

Ultrasensitive detection of genetically modified maize DNA by capillary gel electrophoresis with laser-induced fluorescence using different fluorescent intercalating dyes.

In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.

Bacillus thuringiensis Toxins↗
PubMed2006

New BODIPY derivatives as OFF-ON fluorescent chemosensor and fluorescent chemodosimeter for Cu2+: cooperative selectivity enhancement toward Cu2+.

New 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) derivatives (1 and 2) were synthesized as an "off-on" fluorescent chemosensor and fluorescent chemodosimeter for Cu2+ and Pb2+. Compound 1 displayed selective and large chelation enhanced fluorescence effects with Pb2+ and Cu2+ among the metal ions examined. On the other hand, compound 2, a fluorescent chemodosimeter, effectively recognized Cu2+ via a selective hydrolysis of the acetyl group.

Boron Compounds↗
PubMed1997

Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

Amino Acid Sequence↗
PubMed1991

A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid.

DL-2-Amino-3-(7-methoxy-4-coumaryl)propionic acid, a new fluorescent amino acid (abbreviated to Amp), has been synthesized to provide an alternative to tryptophan in quenched fluorescent peptide substrates for peptidases. The model compound Ac-DL-Amp-NH2 was intensely fluorescent with an excitation maximum at 328 nm and an emission maximum at 392 nm. Fmoc (fluoren-9-ylmethoxycarbonyl)-DL-Amp was made to allow the solid-phase synthesis of Amp-containing peptides by the Fmoc-polyamide method. The peptide derivative Dnp (2,4-dinitrophenyl)-Pro-Leu-Gly-Pro-DL-Amp-D-Lys was cleaved by thimet peptidase at the Leu-Gly bond, with a 20-fold enhancement of fluorescence. The value of kcat./Km for thimet peptidase was 6.7 x 10(5) M-1.s-1, compared with the value of 2.4 x 10(5) M-1.s-1 for the tryptophan-containing analogue, Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys.

Alanine↗
PubMed1998

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence.

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris. HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (lambdamax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Amino Acid Sequence↗
PubMed2000

Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules.

Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the "on-off" blinking observed in the Ser-65 --> Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 micros and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on- and off-times renders a characteristic off-time of 1.6 +/- 0.2 s and allows us to calculate a transition yield of approximately 0.5 x 10(-5) from the emissive to the nonemissive state. The saturation excitation intensity at which on- and off-times are equal is approximately 1.5 kW/cm(2). On the basis of the single-molecule data we calculate an absorption cross section of 6.5 x 10(-17) cm(2) for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level.

Fluorescence↗
PubMed2001

Disappearance of catecholamine fluorescence from the adrenergic nerves in arterial grafts in rats: an experimental fluorescence histochemical study.

The disappearance of catecholamine fluorescence from the noradrenaline-containing sympathetic nerve fibres after arterial transplantation was studied using a femoral artery graft sutured to rat carotid artery. Glyoxylic acid-induced fluorescence was used to demonstrate adrenergic nerves histochemically. At six hours the network of fibres had started to degenerate, and catecholamine fluorescence from the adrenergic nerves had almost completely disappeared within 24 hours of grafting. Control specimens from normal femoral arteries showed a dense network of fluorescent adrenergic nerves. Based on observations of the relatively rapid liberation of catecholamines from the degenerating adrenergic nerves, we suggest that catecholamines liberated from degenerating adrenergic nerves may have an important role in early vasospasm in microvascular and coronary bypass surgery.

Adrenergic Fibers↗
PubMed1982

A new assay method for DNase by fluorescence polarization and fluorescence intensity using DNA-ethidium bromide complex as a sensitive substrate.

The fluorescence polarization method was applied for the determination of DNase-activity using ethidium bromide-labeled DNA as a substrate. This assay was based on a decrease in fluorescence intensity or fluorescence polarization value, which reflected degraded molecular size resulting from the specific cleavage of DNA by the enzyme. DNase activity was quantitatively determined above 0.1 ng/ml of the enzyme. A decrease in fluorescence intensity or polarization value brought about by DNase was inhibited by the specific inhibitors of DNase such as EDTA, pyrophosphate, and thymidine-3',5'-diphosphate. In addition, specificity of the change of polarization value was established using other enzymes. Since this method was simple, rapid and sensitive, it is applicable for both quantitative and qualitative assays as well as for the kinetic analysis of the enzyme.

DNA↗
PubMed2005

Fluorescently tagged canine adenovirus via modification with protein IX-enhanced green fluorescent protein.

Canine adenovirus type 2 (CAV2) has become an attractive vector for gene therapy because of its non-pathogenicity and the lack of pre-existing neutralizing antibodies against this virus in the human population. Additionally, this vector has been proposed as a conditionally replicative adenovirus agent under the control of an osteocalcin promoter for evaluation in a syngeneic, immunocompetent canine model with spontaneous osteosarcoma. In this study, a CAV2 vector labelled with the fluorescent capsid fusion protein IX-enhanced green fluorescent protein (pIX-EGFP) was developed. Expression of the fluorescent fusion-protein label in infected cells with proper nuclear localization, and incorporation into virions, could be detected. The labelled virions could be visualized by fluorescence microscopy; this was applicable to the tracking of CAV2 infection, as well as localizing the distribution of the vector in tissues. Expression of pIX-EGFP could be exploited to detect the replication and spread of CAV2. These results indicate that pIX can serve as a platform for incorporation of heterologous proteins in the context of a canine adenovirus xenotype. It is believed that capsid-labelled CAV2 has utility for vector-development studies and for monitoring CAV2-based oncolytic adenovirus replication.

Adenoviruses, Canine↗
PubMedDate not supplied

Fluorescence resonance energy transfer imaging via fluorescence polarization measurement.

Fluorescence resonance energy transfer (FRET) has become a widely used spectroscopic tool for detecting molecular interactions and molecular proximity in solution, as well as in membranes. On the other hand, fluorescence polarization (FP) is a convenient measure: ratiometric and simple to execute. This work presents a novel methodology for determining energy transfer efficiency (E) via FP measurement. The methodology is based on the fact that a donor's fluorescence lifetime is shortened due to FRET and, consequently, its FP increases. As a model, the present work evaluates the E between fluorescein and rhodamine conjugated ConA attached to the receptors in the lymphocyte membrane. It shows not only that FRET imaging via FP is possible, but also that it is inexpensive, simple to perform, conveniently adaptable to the commonly used fluorescent microscopy, and readily interpretable.

Cell Line↗
PubMed2003

Microplate bioassay for nisin in foods, based on nisin-induced green fluorescent protein fluorescence.

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 microg of nisin in cheese, and 1 microg of nisin per ml in salad dressings.

Animals↗
PubMed1981

Sequential alterations in the nuclear chromatin region during mitosis of the fission yeast Schizosaccharomyces pombe: video fluorescence microscopy of synchronously growing wild-type and cold-sensitive cdc mutants by using a DNA-binding fluorescent probe.

Video-connected fluorescence microscopy was introduced to study the yeast nuclear chromatin region. It was defined as the nuclear area where a DNA-binding fluorescent probe 4',6-diamidino-2-phenylindole specifically bound and fluoresced. The 3-dimensional feature of the mitotic chromatin region was deduced by analysing the successive video images of a cell viewed at different angles. By investigating synchronous culture of the wild-type fission yeast Schizosaccharomyces pombe, we found sequential structural alterations in the chromatin region during mitosis. The steps found include the compaction of the chromatin region from the regular hemispherical form, the formation of a U-shaped intermediate and the rapid segregation into 2 daughter hemispherical forms. Six cs cdc mutants, apparently blocked in mitosis, were observed by fluorescence microscopy. Under the restrictive conditions their chromatin regions exhibited either hemispherical, compact, disk-like, U-shaped or partially segregated chromatin regions. Two mutants showed anomalous nuclear locations. The results of the temperature shift-up experiments of the highly reversible KM52 and KM108 strains supported the above scheme of sequential alterations in the chromatin region.

Ascomycota↗
PubMed2006

Fast and reversible photoswitching of the fluorescent protein dronpa as evidenced by fluorescence correlation spectroscopy.

Controlling molecular properties through photoirradiation holds great promise for its potential for noninvasive and selective manipulation of matter. Photochromism has been observed for several different molecules, including green fluorescent proteins, and recently the discovery of a novel photoswitchable green fluorescent protein called Dronpa was reported. Dronpa displays reversible and highly efficient on/off photoswitching of its fluorescence emission, and reversible switching of immobilized single molecules of Dronpa with response times faster than 20 ms was demonstrated. In this Letter, we expand these observations to freely diffusing molecules by using fluorescence correlation spectroscopy with simultaneous excitation at 488 and 405 nm. By varying the intensity of irradiation at 405 nm, we demonstrate the reversible photoswitching of Dronpa under these conditions, and from the obtained autocorrelation functions we conclude that this photoswitching can occur within tens of microseconds.

Dose-Response Relationship, Radiation↗
PubMed2001

Fluorescence produced by transfection reagents can be confused with green fluorescent proteins in mammalian cells.

The Aequorea victoria green fluorescent protein (GFP) reporter system is a convenient way to monitor gene expression and other cellular functions in mammalian cells. To study gene expression, a GFP-fusion plasmid construct is often transfected into mammalian cells using a variety of methods including calcium phosphate- and liposome-based DNA transfer. Subsequently, the expression of GFP-fusion protein is monitored by fluorescence microscopy or flow cytometry. Here, we report that certain transfection reagents can produce fluorescence that can be detected in a wide range of wavelengths, which can be confused with GFP-fusion protein. The fluorescence false positives can be a problem, particularly when the GFP expression levels are low. To improve the GFP-based detection or screening methods, it is imperative to include an appropriate negative control and to detect GFP using a narrow-wavelength emission filter corresponding to the emission spectrum around the GFP peak.

Animals↗
PubMed1999

Fusion of the binding domain of Raf-1 kinase with green fluorescent protein for activated Ras detection by fluorescence correlation spectroscopy.

Ras proto-oncogenes play a central role in cell proliferation by the regulation of signal transduction pathways from receptors of the outer cell membrane to the nucleus via the activation of transcription factors. Wild-type Ras cycles between the activated GTP-bound and the inactivated GDP-bound state, and the GTPase reaction is a timer for the interaction between Ras-GTP and effector molecules such as Raf-1 protein kinase. Mutations of ras resulting in the loss of the intrinsic GTPase activity result in autonomous proliferation. Mutated Ras is found in a variety of human tumors. Therefore, monitoring of GTP-loaded conformation of Ras related proteins could be utilised in cancer diagnosis. To develop a fluorescence based bioassay we have coupled the gene for the N-terminal Ras binding domain (RBD) of Raf-1 protein kinase with the gene for the green fluorescent protein (GFP). The chimeric fusion protein RBDGFP was identified by immunoblotting and subsequently investigated by fluorescence correlation spectroscopy (FCS), a new analytical technology allowing the measurement of characteristic diffusion times of fluorescently labeled molecules. Molecular interactions increase the molecular weight and influence the diffusion time of RBDGFP. FCS diffusion value of the recombinant protein was in coincidence with the molecular weight of the construct. Fluorimetric measurements of RBDGFP versus GFP showed clearly that the recombinant protein contains functional GFP. Increased FCS transition times indicated the interaction of RBDGFP with its corresponding antibody. Suboptimal binding of the fusion protein to activated Ras, how ever, resulted in a modest influence on the diffusion value. Taken together our rational design and construct shows the way for a ready characterisation of novel GFP-connected fusion proteins employing FCS.

Blotting, Western↗
PubMed2000

Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.

Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and "splinked" ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of the lys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neo gene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile. (Blood. 2000;96:719-726)

Animals↗