Fluorescamine Research Articles

Functional Imaging with Chemically Amplified Resists and Organic Molecules

Chemically amplified resists were tested for preparing universal templates to bind various organic species in micron-scale patterns (functional images). UV exposure and heating of a poly(di-tert-butyl fumarate-co-styrene):onium salt resist produced alternating areas of nonpolar ester and polar acid/anhydride polymers. Various compounds that were fluorescent dyes or amines could then be placed selectively either into or onto exposed or nonexposed areas, according to the nature of solvents and binding species. The ease and extent of immobilization were found to depend on (i) resist composition, (ii) matrix wettability and permeability by a solvent, and (iii) covalent, ionic, dipole−dipole, or other sorbate−matrix binding interactions.

Basal ganglia organization in amphibians: Efferent connections of the striatum and the nucleus accumbens

As a further step in unraveling the organization of the basal ganglia of amphibians, the efferent connections of the striatum and the nucleus accumbens have been studied in the brains of the anurans, Rana perezi and Xenopus laevis, and the urodele, Pleurodeles waltl, by using biotinylated or fluorescent dextran amines as anterograde tracers. A common pattern of efferent connections was observed in both groups of amphibians, but those in the anurans were more elaborate. Striatal efferent fibers were found to reach the lateral and medial amygdala, the anterior and posterior entopeduncular nuclei, several thalamic nuclei, the dorsomedial posterior tubercle, the pretectum, the optic tectum, the torus semicircularis, the pontomesencephalic reticular formation, and the caudal brainstem. Efferent fibers of the nucleus accumbens project to the medial amygdala, the preoptic area, the ventral hypothalamic nucleus, the dorsomedial posterior tubercle, the medial tegmental area, the pontomesencephalic reticular formation, and the raphe. In addition, the study has revealed the existence of intrinsic connections within the ventral telencephalic wall, suggesting a possible further compartmentalization of the amphibian basal forebrain. In conclusion, the results of the present study corroborate the notion that the basal ganglia of amphibians share many features with their presumed homologues in amniotes.

Basal ganglia organization in amphibians: Efferent connections of the striatum and the nucleus accumbens

As a further step in unraveling the organization of the basal ganglia of amphibians, the efferent connections of the striatum and the nucleus accumbens have been studied in the brains of the anurans, Rana perezi and Xenopus laevis, and the urodele, Pleurodeles waltl, by using biotinylated or fluorescent dextran amines as anterograde tracers. A common pattern of efferent connections was observed in both groups of amphibians, but those in the anurans were more elaborate. Striatal efferent fibers were found to reach the lateral and medial amygdala, the anterior and posterior entopeduncular nuclei, several thalamic nuclei, the dorsomedial posterior tubercle, the pretectum, the optic tectum, the torus semicircularis, the pontomesencephalic reticular formation, and the caudal brainstem. Efferent fibers of the nucleus accumbens project to the medial amygdala, the preoptic area, the ventral hypothalamic nucleus, the dorsomedial posterior tubercle, the medial tegmental area, the pontomesencephalic reticular formation, and the raphe. In addition, the study has revealed the existence of intrinsic connections within the ventral telencephalic wall, suggesting a possible further compartmentalization of the amphibian basal forebrain. In conclusion, the results of the present study corroborate the notion that the basal ganglia of amphibians share many features with their presumed homologues in amniotes. J. Comp. Neurol. 380:23–50, 1997. © 1997 Wiley-Liss, Inc.

3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde, a Reagent with Broad Dynamic Range for the Assay of Proteins and Lipoproteins in Solution

This paper describes the application of 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), a sensitive fluorogenic reagent for detection of amines, to the assay of proteins in solution. The sensitivity and dynamic range of CBQCA in the determination of protein concentration is modulated by the number of accessible amines present in the protein assayed. Thus, this method bears the same limitations as other reagents used in fluorogenic assays based on fluorescent amine adducts such as those obtained with fluorescamine oro-phthaldialdehyde, or of other spectrophotometric methods, where the color development is determined by the abundance in the protein of certain amino acids. However, in comparison to other fluorescence-based protein detection methods, CBQCA has proven to be an extremely sensitive reagent with a very broad, essentially linear dynamic range, capable of detecting from 10 ng to 150 μg of protein (in a 100- to 200-μL assay volume). The CBQCA reagent also functions well in the presence of substances, such as lipids, known to interfere in many other protein determination methods.

Glibenclamide in serum: comparison of high-performance liquid chromatography using fluorescence detector and liquid chromatography/mass spectrometry with atmospheric-pressure chemical-ionization (APCI LC/MS).

Comparative studies of high-performance liquid chromatographic and liquid chromatographic/mass spectrometric methods for the quantitative determination of glibenclamide in patient serum are described. The 4-methylcyclohexyl analogue of glibenclamide [N-(4-(beta-5-chloro-2-methoxybenzamidoethyl)benzenesulfonyl-N'-(4-methylcyclohexyl)-urea] is used as internal standard for both methods. After acidification of the sample, the analyte and internal standard are extracted with chloroform. For HPLC analysis, the glibenclamide and internal standard are derivatized to a highly fluorescent amine with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride). The glibenclamide derivative is detected by a fluorescence detector. For APCI LC/MS assay, a derivatization of the analyte is not required and the compounds are measured by selected ion monitoring (SIM). The accuracy, precision, and recovery of the compared methods are presented and discussed.

Spectrofluometric determination of baclofen in tablets with fluorescamine

Spectrofluometric determination of baclofen in tablets with fluorescamine

Acidification of lysosomes and endosomes.

Lysosomes, endosomes, and a variety of other intracellular organelles are acidified by a family of unique proton pumps, termed the vacuolar H(+)-ATPases, that are evolutionarily related to bacterial membrane proton pumps and the F1-F0 H(+)-ATPases that catalyze ATP synthesis in mitochondria and chloroplasts. The electrogenic vacuolar H(+)-ATPase is responsible for generating electrical and chemical gradients across organelle membranes with the magnitude of these gradients ultimately determined by both proton pump regulatory mechanisms and, more importantly, associated ion and organic solute transporters located in vesicle membranes. Analogous to Na+, K(+)-ATPase on the cell membrane, the vacuolar proton pump not only acidifies the vesicle interior but provides a potential energy source for driving a variety of coupled transporters, many of them unique to specific organelles. Although the basic mechanism for organelle acidification is now well understood, it is already apparent that there are many differences in both the function of the proton pump and the associated transporters in different organelles and different cell types. These differences and their physiologic and pathophysiologic implications are exciting areas for future investigation.

Identification of Factor‐XIIIa‐Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100-kDa propeptide, called the propolypeptide of von Willebrand factor (pp-vWF), is generated during biosynthesis. We found that pp-vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311-12316]. As such, it could form cross-linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp-vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861-867]. In this work, we identified the Gln residues in pp-vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme-reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti-dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp-vWF, only four could be modified in the factor-XIIIa-catalyzed reaction.

Automated Subpicomole Protein Sequencing Using an Alternative Postcleavage Conversion Chemistry

An automated subpicomole sequencing method is described. It is based on a chemistry that generates easily detectable amino acid derivatives from the postcleavage products of the classical Edman degradation. These products, which include the amino acid anilinothiazolinone (ATZ), phenyithiocarbamyl (PTC), and phenyithiohydantoin (PTH), are converted to a homogeneous preparation of ATZ that can react with sensitivity-enhancing compounds such as fluorescent amines. The method is demonstrated with a protein of known sequence (α-lactalbumin) and appears to offer a significant improvement over current high-sensitivity protein sequencing strategies. Modifications to the sequencer hardware or to the Edman degradation chemistry are not required.

Early development of dorsal column-medial lemniscal projections in the clawed toad, Xenopus laevis

In Xenopus laevis fluorescent dextran amines were applied to study the development of the dorsal column-medial lemniscal projection: rhodamine dextran amine was applied at the mesodiencephalic border to retrogradely label the cells of origin of the medial lemniscus in the dorsal column nucleus (DCN); fluorescein dextran amine to the spinal cord to anterogradely label the primary afferent projections to the DCN. The first mesodiencephalic projections were found at stage 51, i.e. almost immediately after spinal afferent fibers had reached the DCN.

Liquid chromatographic and spectrofluorimetric determination of aspartame and glutamate in foodstuffs following fluorescamine fluorigenic labelling

A spectrofluorimetric method for determining aspartame (AS) and sodium glutamate (GL) based on labelling with fluorescamine (FC) was developed. Mixtures of AS and GL were analysed by isodifferential synchronous derivative spectrofluorimetry. R.S.D.s of 6.4% and 3.0% were obtained by the individual methods. Detection limits of 0.03 and 0.1 μg ml -1 were obtained for AS and GL, respectively. On the basis of the FC derivatization procedure, a liquid chromatographic method to determine AS amd GL was developed. R.S.D.s of 0.62 and 0.34% were obtained with fluorimetric detection and 2.26 and 3.14% with photometric detection at 395 nm. Detection limits ere 0.04 and 0.02 μg ml -1 (spectrofluorimetric detection) and 0.2 and 0.1 μg ml -1 (spectrophotometric detection) for AS and GL, respectively. These methods were applied to the determination of AS in carbonated soft drinks and GL analyses in commercial dried soups.

Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group

The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.

Pharmacologic evaluation of factor XIIIa*-like enzyme activity in equine plasma as a potential therapeutic avenue for the inhibition of fibrinous tissue

Summary Several pharmaceutical compounds were evaluated for their ability to selectively inhibit activated coagulation factor-XIII-like enzyme activity (eg, Xllla*) in pooled equine plasma. Presence of coagulation factor-XIIIa*-like enzyme activity in plasma was established by assay procedures involving incorporation of the fluorescent amine compound, monodansylcadaverine, into purified casein, which served as a protein substrate. Pharmaceuticals inhibitory to coagulation factor-XIIIa*-like enzyme activity were recognized by plasma gel formation of high spectrophotometric transmittance (transparency), solubility of transparent fibrin gels in concentrated urea solution, in conjunction with simultaneous depletion of native fibrinogen fractions, and production of fibrin monomer. Compounds acting primarily as anticoagulants were recognized by lack of plasma gel formation, but retaining high spectrophotometric transmittance and no detectable depletion of native fibrinogen fractions. Compounds failing to inhibit either thrombin-mediated fibrinogen-fibrin transformation (ie, coagulation) or coagulation factor-XIIIa*-like enzyme activity were recognized by opaque plasma gels caused by fibrin polymerization, low spectrophotometric transmittance values, and coinciding with depletion of native fibrinogen fractions. Pharmaceuticals capable of exerting selective inhibition of coagulation factor-XIIIa*-like enzyme activity were further classified as competitive inhibitors of phase 1 (carbamide) or phase 2 (terminal amine) of the transglutamination process.

Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies

Fluorescent dextran amines have recently been reported to be useful for anterograde pathway tracing. However, fluorescent markers are not always ideal for detailed mapping studies. We therefore evaluated the efficacy of a biotinylated dextran amine (BDA) for anterograde labeling in several different preparations. BDA was visualized with an avidin biotinylated HRP (ABC) procedure followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. After iontophoretic injections of BDA into neocortex-like telencephalic regions in pigeons or into visual or somatosensory cortex in rats, there was excellent and abundant labeling of axons and terminals in forebrain, midbrain and hindbrain target areas with 1-week survival times. Large pressure injections of BDA into the avian telencephalon were also found to result in extensive anterograde labeling. We then carried out a series of studies using 2-color DAB double-labeling to determine effective approaches for combining BDA labeling with other labeling methods. Using an isolated embryonic chick spinal cord-hindlimb preparation, we combined BDA labeling with another anterograde labeling method to differentially label two sets of projections. In these studies, sensory neuron and motoneuron projections into the limb from the same segmental level, or motoneuron projections into the limb from two separate segments were differentially labeled by using HRP (visualized first with a blue/black metal-DAB reaction) and BDA (visualized second with a brown DAB reaction). In other double-labeling studies, we combined BDA labeling of axons and terminals with immunohistochemical labeling of neurons. In these experiments, telencephalic neurons in pigeons or rats were labeled immunohistochemically for parvalbumin or substance P (using a brown DAB reaction) and BDA-labeled axons were labeled blue/black (using a metal-intensified DAB reaction). Double-labeling was successful regardless of whether the entire immunohistochemical labeling procedure preceded or followed the BDA labeling procedure. Together, these studies show that BDA is effective for anterograde pathway tracing and can be used in double-label studies with other labeling methods.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl- l-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent K m of 34.7 ± 2.4 m m was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, α-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.

Merkel cells in vitro: Production of nerve growth factor and selective interactions with sensory neurons

A method has been developed for obtaining mixed primary cultures of dissociated epidermis enriched in Merkel cells. Merkel cells obtained from embryonic rat buccal pads were grown in serum-free medium and identified in vitro using a variety of histological and immunohistochemical markers. Quinacrine, a fluorescent amine, which has been used to identify Merkel cells in situ, labeled a morphologically distinct population of cells in vitro. Cells labeled with quinacrine had a large, phase bright nucleus with prominent nucleoli, surrounded by a phase dark perinuclear ring. Antibodies directed against neuron-specific enolase, another marker for Merkel cells in situ, and antibodies against a well-characterized neuroendocrine vesicle antigen also labeled this population of quinacrine fluorescent cells. Electron microscopic examination of our cultures indicated that cells containing characteristic features of Merkel cells including cytoplasmic dense-cored granules were present. A small but significant increase in the number of Merkel cells was observed over time in culture. Merkel cells supported the survival and outgrowth of both trigeminal ganglion sensory neurons and sympathetic neurons from the superior cervical ganglion in serum-free medium in the absence of exogenous nerve growth factor (NGF). Immunoblots probed with antibodies directed against NGF demonstrated that NGF was present in the medium taken from these cultures. NGF-like immunoreactivity colocalized to cells containing quinacrine fluorescence in situ and in vitro. Addition of antibodies directed against NGF to cocultures of Merkel cells and neurons decreased survival of sympathetic neurons by 90% and decreased survival of sensory neurons by 60%. These results suggest that Merkel cells are capable of providing trophic support for their normal complement of sensory neurons by producing NGF. Selective recognition of these targets was studied in vitro by characterizing the interactions between Merkel cells and growth cones from sensory or sympathetic neurons using both time-lapse videomicroscopy and standard morphometry of fixed cocultures. The majority of trigeminal ganglion sensory neurons (approximately 60%) extended growth cones onto clusters of Merkel cells. Neurites which contacted clusters of Merkel cells were significantly more highly branched than those growing on collagen. In contrast, the majority of sympathetic neurons (greater than 90%) failed to grow onto Merkel cells. Growth cones of sympathetic neurons often “collapsed” and retracted when contact was made with a cluster of Merkel cells. Fixation of Merkel cells with paraformaldehyde prior to coculture did not affect this difference between sensory and sympathetic neurite extension onto the Merkel cells. However, prior fixation of Merkel cells eradicated the apparent Merkel cell-induced branching of sensory neurites. These results suggest that innervation of Merkel cells in vivo may be limited to sensory neurons, in part, by molecules on the surface of these target cells. Furthermore, the extensive arborization of sensory neurons which occurs in the vicinity of these targets in vivo may be due to the production of a target-derived agent.

Delayed intermolecular ??-chain cross-linking by factor XIIIa in fibrinogen Asahi characterized by a ??-Met-310 to Thr substitution with an N-glycosylated ??-Asn-308

In an abnormal fibrinogen with gamma-Met-310 to Thr substitution accompanied by an extra oligosaccharide attached to gamma-Asn-308, factor-XIIIa-mediated intermolecular gamma-dimer formation of fibrin was found to be markedly delayed. The delayed gamma-dimer formation was not due to impaired fibrin polymerization because the fibrinogen gamma-chain also failed to be efficiently cross-linked by factor XIIIa. Since fluorescent amine was normally incorporated into the abnormal gamma-chain by factor XIIIa, we conclude that the abnormal molecules were unable to align their gamma-chains in an anti-parallel fashion because of inaccessibility of the molecules with a profoundly perturbed conformation near the carboxyl terminal region of the gamma-chain included in the D domain.

Differential Inhibition of Tonoplast H+-ATPase Activities by Fluorescamine and Its Derivatives

Corn (Zea mays L.) root tonoplast vesicles were treated with the primary-amine specific reagent, fluorescamine (FL). Modification by FL caused a differential inhibition to the coupled activities of tonoplast H(+)-ATPase. Within the range of 0 to 5 micromoles of FL per milligram of protein, the proton pumping rate was significantly reduced but ATP hydrolysis was only slightly affected. Yet, the membrane H(+) leakage during the pumping stage increased only slightly. FL treatment resulted in (a) a decrease in amine containing phospholipids and (b) an insertion of multiple H-bonding moieties into the membrane. To test which of these two possible effects were responsible for inhibition, FL derivatives of benzylamine, butylamine, and phenylalanine were synthesized. It was found that the acyclic derivatives with high H-bonding potential at concentrations of 10 micromolar inhibited proton pumping by 50% without a significant effect on ATP hydrolysis. Cyclic derivatives were largely ineffectual. Proton leakage during pumping was not affected by these acyclic modifiers. Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane.

Quantitative image analysis of fluorescent biogenic amines in tissue

Quantitative image analysis of fluorescent biogenic amines in tissue

A discrete projection of the sacculus and lagena to a distinct brainstem nucleus in a catfish

The projection of individual inner ear maculae of the catfish, Ancistrus spec. were labelled with horseradish peroxidases (HRP) or fluorescent dextran amines. In ostariophysans, fibres which innervate epithelia known or assumed to be sensitive to the pressure component of aquatic sound, i.e. acoustic fibres which come from the sacculus and lagena, have projections separate from those of the utriculus and canal organs. In Ancistrus afferents of sacculus and lagena terminate in an area which is distinct based on its cytoarchitecture. This area, previously believed to be a secondary auditory nucleus, is therefore an identifiable primary auditory nucleus in a subtetrapod vertebrate. This is different from other pressure sensitive teleosts (clupeids, osteoglossomorphs) in which acoustic fibres also terminate separately from vestibular fibres, but within parts of a larger cytoarchitectonic entity.