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1.
Cytometry A ; 83(8): 752-62, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23765858

ABSTRACT

We have recently added three new fluorophores-BD Horizon™ V450, BD Horizon V500, and BD Horizon V550 (V450, V500, and V550; BD Biosciences, San Jose, CA) to our existing AmCyan product, forming a group of four violet-excitable dyes from which we have produced functional antibody conjugates. These conjugates, with emission maxima that range from 450 to 535 nm, are compatible with multilaser flow cytometry (FCM) and can be used for polychromatic FCM in three-color or two-color combinations; in fact, V500 fills a spectral opening that has thus far not been exploited by other manufacturers of FCM reagents. We here report that conjugates based on BD Horizon dyes performed well within a useful sensitivity range, established by testing a representative group of violet-excitable FCM reagents currently available, and that V500 has better compatibility with FITC in multicolor applications than does AmCyan.


Subject(s)
Flow Cytometry/methods , Fluorescent Dyes/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Fluorescent Antibody Technique , Humans , Hydrogen-Ion Concentration , Immunologic Memory , Phosphorylation , Protein Processing, Post-Translational , Staining and Labeling , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
2.
Cytometry A ; 81(7): 627-34, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22674817

ABSTRACT

Here we report a new variant of AmCyan fluorescent protein that has been specifically designed for multicolor cell analysis. AmCyan is one of the existing violet fluorochromes for use in flow cytometers equipped with a violet (405 nm) laser. It is also widely used as a label in fluorescent spectroscopy. Limitations on its use are due to the significant AmCyan fluorescence spillover into the FITC detector, due to excitation of AmCyan by the blue (488 nm) laser. In order to resolve this problem, we modified the excitation profile of AmCyan. The new fluorescent protein that we developed, AmCyan100, has an emission profile similar to AmCyan with an emission maximum at 500 nm, but its excitation maximum is shifted to 395 nm, which coincides more closely with the violet laser line and decreases the excitation with the blue laser, thus reducing the spillover observed with the original AmCyan. Moreover, this new protein has a Stokes shift of more than 100 nm compared to the Stokes shift of 31 nm in its precursor. Our data also suggests that AmCyan100-mAb conjugates have brightness similar to AmCyan-mAb conjugates. In summary, AmCyan100 conjugates have minimum spillover into the FITC detector, and can potentially replace existing AmCyan conjugates in multicolor flow cytometry without any changes in instrumental setup and existing reagent panel design.


Subject(s)
Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Amino Acid Substitution , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cloning, Molecular , Cytokines/metabolism , Escherichia coli , Fixatives/chemistry , Flow Cytometry , Fluorescence , Fluorescent Antibody Technique, Direct , Formaldehyde/chemistry , Green Fluorescent Proteins/genetics , Humans , Mutagenesis , Polymers/chemistry , Protein Engineering , Tissue Fixation
3.
Anal Chem ; 83(18): 7109-14, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21846137

ABSTRACT

When proteins are conjugated to fluorescent organic dyes, fluorescence emission of the dye molecules is usually decreased, sometimes up to 50-70%. This quenching phenomenon has been acknowledged for decades, but as yet, there are no simple, practical methods to control the fluorescence of dyes conjugated to proteins, especially for dyes conjugated to immunoglobulins. Here, we report that the addition of (2-hydroxypropyl)-ß-cyclodextrin (HPßCD) to dye-antibody conjugates can increase fluorescence up to 2.5-fold in cell imaging and flow analysis. This method may be an effective way to increase the sensitivity of detection of fluorescent organic labels used in immunology, histochemistry, and cell biology.


Subject(s)
Flow Cytometry/methods , Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , beta-Cyclodextrins/chemistry , 2-Hydroxypropyl-beta-cyclodextrin , Antibodies/chemistry , Antibodies/immunology , Humans , Immunoglobulins/chemistry , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology
4.
Endocrinology ; 150(9): 4154-62, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19556422

ABSTRACT

Human 17beta-hydroxysteroid dehydrogenase types 1 and 2 (17betaHSD1 and 17betaHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17betaHSD1 and 17betaHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2/E1 ratio achieved by 17betaHSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 after mutagenesis of R38, which interacts with the 2'-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP(+) ratio. The shift to E2 oxidation parallels changes in apparent K(m) values for purified 17betaHSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17betaHSD1) and changes in intracellular cofactor ratios do not alter the greater than 90:10 E1/E2 ratio catalyzed by 17betaHSD2, and these mutations lower the apparent K(m) of recombinant 17betaHSD2 for NADP(H) only less than 3-fold. We conclude that the equilibrium E1/E2 ratio maintained by human 17betaHSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP(+) ratios. In contrast, the preference of 17betaHSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding and thus support E2 oxidation by 17betaHSD2 in intact cells.


Subject(s)
17-Hydroxysteroid Dehydrogenases/metabolism , Estradiol Dehydrogenases/metabolism , 17-Hydroxysteroid Dehydrogenases/genetics , Amino Acid Sequence , Amino Acid Substitution , Cell Line , Cloning, Molecular , Estradiol/metabolism , Estrone/metabolism , Humans , Kidney/embryology , Kidney/enzymology , Kinetics , Molecular Sequence Data , NADP/metabolism , Sequence Alignment
5.
Anal Biochem ; 386(2): 262-9, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19135024

ABSTRACT

In our search for new violet-excitable dyes with improved photophysical and photochemical properties, we examined several halogen-substituted hydroxycoumarins and found that chlorinated derivatives are at least as bright as their fluorinated analogs. A monochlorinated hydroxycoumarin was found to have a high quantum yield (approximately 0.98), and human leucocyte-specific monoclonal antibodies (CD3, CD4, and CD45) conjugated with this dye exhibited reliable performance in flow cytometry assays. Additional studies were performed, with BD Horizon V450-antibody conjugates being included in eight-color cocktails aimed at subsetting lymphocytes and myeloid cells. Such cocktails can frequently be unstable due to the tendency of one or more components to lose structural integrity, photobleach, or develop unwanted affinities for another component. However, the cocktails employed in this study enabled several different applications to be run and established that multicolor reagent mixtures containing V450-antibody conjugates are functional and stable.


Subject(s)
Coumarins/chemistry , Fluorescent Dyes/chemistry , Umbelliferones/chemistry , CD3 Complex/chemistry , CD3 Complex/metabolism , CD4 Antigens/chemistry , CD4 Antigens/metabolism , Cells, Cultured , Flow Cytometry , Humans , Hydrogen-Ion Concentration , Leukocyte Common Antigens/chemistry , Leukocyte Common Antigens/metabolism , Photobleaching , Spectrometry, Fluorescence
6.
Nat Struct Mol Biol ; 12(11): 1017-8, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16228007

ABSTRACT

Munc13 proteins are essential in neurotransmitter release, controlling the priming of synaptic vesicles to a release-ready state. The sequences responsible for this priming activity are unknown. Here we identify a large alpha-helical domain of mammalian Munc13-1 that is autonomously folded and is sufficient to rescue the total arrest in neurotransmitter release observed in hippocampal neurons lacking Munc13s.


Subject(s)
Exocytosis/physiology , Gene Expression , Hippocampus/metabolism , Nerve Tissue Proteins/genetics , Neurons/metabolism , Animals , Chromatography, Gel , Computational Biology , Excitatory Postsynaptic Potentials , Exocytosis/genetics , Mice , Mice, Knockout , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Neurotransmitter Agents/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Folding , Protein Structure, Secondary/genetics , Protein Structure, Tertiary , Rats , Structure-Activity Relationship , Sucrose/pharmacology
7.
Biochemistry ; 44(4): 1089-96, 2005 Feb 01.
Article in English | MEDLINE | ID: mdl-15667202

ABSTRACT

Protein kinase C (PKC) isozymes and other receptors of diacylglycerol (DAG) bind to this widespread second messenger through their C(1) domains. These alternative DAG receptors include munc13-1, a large neuronal protein that is crucial for DAG-dependent augmentation of neurotransmitter release. Whereas the structures of several PKC C(1) domains have been determined and have been shown to require little conformational changes for ligand binding, it is unclear whether the C(1) domains from other DAG receptors contain specific structural features with key functional significance. To gain insight into this question, we have determined the three-dimensional structure in solution of the munc13-1 C(1) domain using NMR spectroscopy. The overall structure includes two beta-sheets, a short C-terminal alpha-helix, and two Zn(2+)-binding sites, resembling the structures of PKC C(1) domains. However, the munc13-1 C(1) domain exhibits striking structural differences with the PKC C(1) domains in the ligand-binding site. These differences result in occlusion of the binding site of the munc13-1 C(1) domain by a conserved tryptophan side chain that in PKCs adopts a completely different orientation. As a consequence, the munc13-1 C(1) domain requires a considerable conformational change for ligand binding. This structural distinction is expected to decrease the DAG affinity of munc13-1 compared to that of PKCs, and is likely to be critical for munc13-1 function. On the basis of these results, we propose that augmentation of neurotransmitter release may be activated at higher DAG levels than PKCs as a potential mechanism for uncoupling augmentation of release from the multitude of other signaling processes mediated by DAG.


Subject(s)
Diglycerides/antagonists & inhibitors , Diglycerides/chemistry , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/chemistry , Amino Acid Sequence , Animals , Binding Sites/genetics , Conserved Sequence , Crystallography, X-Ray , Diglycerides/physiology , Isoenzymes/chemistry , Isoenzymes/metabolism , Ligands , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Kinase C/chemistry , Protein Kinase C/metabolism , Protein Kinase C-delta , Protein Structure, Tertiary/genetics , Rats , Second Messenger Systems , Tryptophan/chemistry
9.
Arch Biochem Biophys ; 418(2): 151-60, 2003 Oct 15.
Article in English | MEDLINE | ID: mdl-14522586

ABSTRACT

The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities. hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity. hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity. Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold. Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone. We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17. The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.


Subject(s)
5-alpha-Dihydroprogesterone/chemistry , 5-alpha-Dihydroprogesterone/metabolism , Androgens/biosynthesis , Pregnanolone/chemistry , Pregnanolone/metabolism , Steroid 17-alpha-Hydroxylase/chemistry , Steroid 17-alpha-Hydroxylase/metabolism , Gonads/metabolism , Humans , Hydroxylation , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Steroids/chemistry , Steroids/metabolism , Substrate Specificity
10.
Endocrinology ; 144(2): 575-80, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12538619

ABSTRACT

The synthetic pathway by which 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20-40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [(3)H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5alpha-adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5alpha-pregnane-3alpha,17alpha-diol-20-one (5alpha-pdiol) and androsterone as intermediates. Formation of 5alpha-adiol from both [(3)H]testosterone and [(3)H]progesterone was blocked by the 5alpha-reductase inhibitor 4MA. The addition of nonradioactive 5alpha-pdiol blocked the conversion of [(3)H]progesterone to 5alpha-adiol, and [(3)H]5alpha-pdiol was efficiently converted to androsterone and 5alpha-adiol. We conclude that expression of steroid 5alpha-reductase in the developing wallaby testes allows formation of 5alpha-reduced androgens by a pathway that does not involve testosterone as an intermediate.


Subject(s)
Androstane-3,17-diol/biosynthesis , Macropodidae/metabolism , Pregnanediol/metabolism , Testis/metabolism , Age Factors , Animals , Female , Male , Pregnanediol/analogs & derivatives , Progestins/pharmacokinetics , Testis/growth & development , Testosterone/pharmacokinetics , Tritium
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