Wyniki wyszukiwania dla: Richard P. Haugland

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Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator

Andrei L. Kindzelskii, Imran Amhad, Donald Keller, M.-J. Zhou, więcej

Histochemistry and Cell Biology > 2004 > 121 > 4 > 299-310

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are...

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Rational design and synthesis of a novel class of highly fluorescent rhodamine dyes that have strong absorption at long wavelengths

Jixiang Liu, Zhenjun Diwu, Wai-Yee Leung, Yixin Lu, więcej

Tetrahedron Letters > 2003 > 44 > 23 > 4355 - 4359

A novel class of strongly fluorescent rhodamine dyes were designed and synthesized by extending the π conjugation of chromophore with limited flexibility. These dyes were shown to have longer absorption in the range of 581 to 631 nm with quantum yields between 0.64 and 0.89.

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Assaying the activities of Thermomonospora fusca E ₅ and Trichoderma reesei CBHI cellulase bound to polystyrene

Sasithorn Kongruang, Michelle K. Bothwell, Joseph McGuire, Mingjie Zhou, więcej

Enzyme and Microbial Technology > 2003 > 32 > 5 > 539-545

The enzymatic activities of adsorbed Thermomonospora fusca E ₅ and Trichoderma reesei CBHI cellulases were investigated using fluorescence techniques. Cellulases were allowed to contact hydrophobic polystyrene surfaces under conditions of different solution concentrations and adsorption times. Each of these variables is known to have an effect on enzyme structure and activity at an interface...

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Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation

James D. Hirsch, Leila Eslamizar, Brian J. Filanoski, Nabi Malekzadeh, więcej

Analytical Biochemistry > 2002 > 308 > 2 > 343-357

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive...

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Sensitive determination of cell number using the CyQUANT(R) cell proliferation assay

Laurie J. Jones, Matthew Gray, Stephen T. Yue, Richard P. Haugland, więcej

Journal of Immunological Methods > 2001 > 254 > 1-2 > 85-98

We describe here the development and characterization of the CyQUANT(R) cell proliferation assay, a highly sensitive, fluorescence-based microplate assay for determining numbers of cultured cells. The assay employs CyQUANT GR dye, which produces a large fluorescence enhancement upon binding to cellular nucleic acids that can be measured using standard fluorescein excitation and emission wavelengths...

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Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy

Chii-Shiarng Chen, Wan-Nan U. Chen, Mingjie Zhou, Seksiri Arttamangkul, więcej

Journal of Biochemical and Biophysical Methods > 2000 > 42 > 3 > 137-151

Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity...

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A Luminescent Ruthenium Complex for Ultrasensitive Detection of Proteins Immobilized on Membrane Supports

Kiera Berggren, Thomas H. Steinberg, Wendy M. Lauber, James A. Carroll, więcej

Analytical Biochemistry > 1999 > 276 > 2 > 129-143

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of...

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Fluorogenic Substrates Based on Fluorinated Umbelliferones for Continuous Assays of Phosphatases and β-Galactosidases

Kyle R. Gee, Wei-Chuan Sun, Mahesh K. Bhalgat, Rosalyn H. Upson, więcej

Analytical Biochemistry > 1999 > 273 > 1 > 41-48

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK_a ≈ 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with...

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7-Amino-4-methyl-6-sulfocoumarin-3-acetic acid: a novel blue fluorescent dye for protein labeling

Wai-Yee Leung, Patty A. Trobridge, Rosaria P. Haugland, Richard P. Haugland, więcej

Bioorganic & Medicinal Chemistry Letters > 1999 > 9 > 15 > 2229-2232

7-Amino-4-methyl-6-sulfocoumarin-3-acetic acid (AMCA-S, also called Alexa ^T ^M 350) 2 was synthesized as a new water-soluble blue fluorescent dye for protein labeling. Compared with its nonsulfonated counterpart (AMCA) 1 the new dye gave significantly higher fluorescence quantum yields on proteins.

artykuł

A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells

Zhenjun Diwu, Chii-Shiarng Chen, Cailan Zhang, Dieter H Klaubert, więcej

Chemistry & Biology > 1999 > 6 > 7 > 411-418

Background: Ratio imaging has received intensive attention in the past few decades. The growing potential of ratio imaging is significantly limited, however, by the lack of appropriate fluorescent probes, for acidic organelles in particular. The classic fluorescent dyes (such as fluoresceins, rhodamines and coumarins) are not suitable for studying acidic organelles (such as lysosomes) because their...

artykuł

5-(Pentafluorobenzoylamino)fluorescein: A Selective Substrate for the Determination of Glutathione Concentration and GlutathioneS-Transferase Activity

Seksiri Arttamangkul, Mahesh K. Bhalgat, Rosaria P. Haugland, Zhenjun Diwu, więcej

Analytical Biochemistry > 1999 > 269 > 2 > 410-417

5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule was synthesized to improve the detection limits and specificity of the assays for glutathioneS-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated...

artykuł

Synthesis of Novel Fluorinated Coumarins: Excellent UV-Light Excitable Fluorescent Dyes

Wei-Chuan Sun, Kyle R. Gee, Richard P. Haugland

Bioorganic & Medicinal Chemistry Letters > 1998 > 8 > 22 > 3107-3110

Two new fluorinated fluorescent dyes, 6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue ^T ^M ) and 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue ^T ^M ), exhibit excellent photophysical properties among a series of novel fluorinated 7-hydroxycoumarins. Most of these fluorinated coumarins have quantum yields (0.63 to 0.89) equal to or higher than that of the parent...

artykuł

Two Fluorometric Approaches to the Measurement of Dextranase Activity

Mingjie Zhou, Cailan Zhang, Rosalyn H. Upson, Richard P. Haugland

Analytical Biochemistry > 1998 > 260 > 2 > 257-259

artykuł

Oscillatory Pericellular Proteolysis and Oxidant Deposition During Neutrophil Locomotion

Andrei L. Kindzelskii, M.-J. Zhou, Richard P. Haugland, Laurence A. Boxer, więcej

Biophysical Journal > 1998 > 74 > 1 > 90-97

To better understand the mechanism of leukocyte migration in complex environments, model extracellular matrices were prepared using gelatin, Hanks’ solution, Bodipy-BSA (fluorescent upon proteolysis), and dihydrotetramethylrosamine or hydroethidine (fluorescent upon oxidation). Using quantitative microfluorometry, neutrophil-mediated extracellular pulses of reactive oxygen metabolites (ROMs) and pericellular...

artykuł

A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases

Mingjie Zhou, Zhenjun Diwu, Nataliya Panchuk-Voloshina, Richard P. Haugland

Analytical Biochemistry > 1997 > 253 > 2 > 162-168

The enzymatic determination of hydrogen peroxide can be accomplished with high sensitivity and specificity usingN-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and chemically stable fluorogenic probe for the enzymatic determination of H₂O₂. Enzyme-catalyzed oxidation of Amplex Red, which is a colorless and nonfluorescent derivative of dihydroresorufin, produces highly fluorescent...

artykuł

Quenched BODIPY Dye-Labeled Casein Substrates for the Assay of Protease Activity by Direct Fluorescence Measurement

Laurie J. Jones, Rosalyn H. Upson, Rosaria P. Haugland, Nataliya Panchuk-Voloshina, więcej

Analytical Biochemistry > 1997 > 251 > 2 > 144-152

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence...

artykuł

Characterization of PicoGreen Reagent and Development of a Fluorescence-Based Solution Assay for Double-Stranded DNA Quantitation

Victoria L. Singer, Laurie J. Jones, Stephen T. Yue, Richard P. Haugland

Analytical Biochemistry > 1997 > 249 > 2 > 228-238

A sensitive assay for detecting double-stranded (ds) DNA in solution is described. This assay employs a new dye, PicoGreen dsDNA quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The brightness of this reagent is due to its high quantum yield (~0.5, bound to ds calf thymus DNA) and large molar extinction coefficient (~70,000 cm ^- ¹ m ^- ¹ ...

artykuł

Fluorescent Molecular Probes I. The Synthesis and Biological Properties of an ELF β-glucuronidase Substrate That Yields Fluorescent Precipitates at the Enzymatic Activity Sites

Diwu Zhenjun, Lu Yixin, Rosalyn H. Upson, Zhou Mingjie, więcej

Tetrahedron > 1997 > 53 > 21 > 7159-7164

A novel quinazolinone-based fluorogenic β-glucuronidase substrate has been synthesized and evaluated. In this protocol, a partially protected salicyl glucuronide is oxidatively condensed with anthranilamide to afford the required quinazolinonyl glucuronic acid methyl ester that is readily deprotected to yield the desired glucuronide, known as ELF -97 glucuronide. The glucuronide is a sensitive...

artykuł

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

Wendy W. You, Rosaria P. Haugland, Diane K. Ryan, Richard P. Haugland

Analytical Biochemistry > 1997 > 244 > 2 > 277-282

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...

artykuł

Fluorescent assay for bacterial gram reaction

Bruce L Roth, Paul Millard, Stephen Yue, K Sam Wells, więcej

Biotechnology Advances > 1997 > 15 > 2 > 411

The invention relates to a method of analyzing a sample thought to contain bacteria using an aqueous solution comprising one or more fluorescent dyes: a fluorescent dye of formula I, a fluorescent dye of formula II, a fluorescent dye of formula III, and a fluorescent dye of formula IV. Each of the dyes differ each from the other in their affinity for nucleic acids and in their spectral response to...

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