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Biomedical subjects

I Bronstein

Publications and source records attributed to I Bronstein.

At least 19 recordsLinked to original sources
PubMed2003

Protein-ligand and protein-protein interactions studied by electrospray ionization and mass spectrometry.

Electrospray ionization has made possible the transference of non-covalently bound complexes from solution phase to high vacuum. In the process, a complex acquires a net charge and becomes amenable to measurement by MS. FTICR (Fourier-transform ion cyclotron resonance) MS allows these ions to be measured with sufficiently high resolution for the isotopomers of complexes of small proteins to be resolved from each other (true for complexes up to about 100 kDa for the most powerful FTICR instruments), which is of crucial significance in the interpretation of spectra. Results are presented for members of the S100 family of proteins, demonstrating how non-covalently bound complexes can be distinguished unambiguously from covalently bound species. Consideration relevant both to determination of binding constants in solution from the gas-phase results and to the elucidation of protein folding and unfolding in solution are discussed. The caveats inherent to the basic approach of using electrospray and MS to characterize protein complexes are weighed and evaluated.

Ligands↗
PubMed2000

Oligomeric forms of the metastasis-related Mts1 (S100A4) protein stimulate neuronal differentiation in cultures of rat hippocampal neurons.

Neuronal differentiation and axonal growth are controlled by a variety of factors including neurotrophic factors, extracellular matrix components, and cell adhesion molecules. Here we describe a novel and very efficient neuritogenic factor, the metastasis-related Mts1 protein, belonging to the S100 protein family. The oligomeric but not the dimeric form of Mts1 strongly induces differentiation of cultured hippocampal neurons. A mutant with a single Y75F amino acid substitution, which stabilizes the dimeric form of Mts1, is unable to promote neurite extension. Disulfide bonds do not play an essential role in the Mts1 neuritogenic activity. Mts1-stimulated neurite outgrowth involves activation of phospholipase C and protein kinase C, depends on the intracellular level of Ca(2+), and requires activation of the extracellular signal-regulated kinases (ERKs) 1 and 2.

Animals↗
PubMed2000

Heterocomplex formation between metastasis-related protein S100A4 (Mts1) and S100A1 as revealed by the yeast two-hybrid system.

S100A4 (Mts1) is a Ca(2+)-binding protein of the S100 family. This protein plays an important role in promoting tumor metastasis. In order to identify S100A4 interacting proteins, we have applied the yeast two-hybrid system as an in vivo approach. By screening a mouse mammary adenocarcinoma library, we have demonstrated that S100A4 forms a heterocomplex with S100A1, another member of the S100 family. The non-covalent heterodimerization was confirmed by fluorescence spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Mutational analysis revealed that replacement of Cys(76) and/or Cys(81) of S100A4 by Ser abolishes the S100A4/S100A1 heterodimerization, but does not affect the S100A4 homodimerization in vivo.

Animals↗
PubMed2000

Immunoassay protocol for quantitation of protein kinase activities.

Quantitation of at least two orders of magnitude of kinase enzyme concentration is achieved with detection of less than 0.1 U/well of src kinase activity (Fig. 3). A comparison between a sequential protocol, in which biotinylated peptide substance is captured prior to incubation with the kinase enzyme, and a simultaneous protocol, in which peptide capture and the kinase reaction proceed concurrently, demonstrates that the simpler simultaneous protocol provides similar detection sensitivity. these have also been demonstrated with 0.1 microM peptide substrate in a protein kinase A assay.5 Quantitation of protein kinase activity with chemiluminescent detection has been demonstrated with several different protein kinases, including both tyrosine and serine/threonine kinases.5 An immunoassay format provides high sensitivity and can be performed under conditions that most closely mimic physiological substrate and ATP concentrations with chemiluminescent detection. This assay format is also automated easily for use in high-throughput screening.

Immunoassay↗
PubMed2000

Chemiluminescent immunodetection protocols with 1,2-dioxetane substrates. 4.

Chemiluminescent 1,2-dioxetane enzyme substrates provide a highly sensitive and versatile detection method for immunoblots and other membrane-based detections. 1,2-Dioxetane substrates, coupled with either alkaline phosphatase or beta-galactosidase enzyme labels, generate glow light emission kinetics, with a signal duration that is significantly longer than most enhanced luminol/horseradish peroxidase chemiluminescent detection systems. The long-lived, high-intensity light signal is ideal for imaging using a variety of formats, including X-ray film, photographic film, chemiluminescence phosphor imaging screens, and the rapidly expanding selection of camera imaging systems.

Blotting, Western↗
PubMed2000

Chemiluminescent reporter gene assays with 1,2-dioxetane enzyme substrates.

1,2-Dioxetane chemiluminescent substrates provide highly sensitive, quantitative detection with simple, rapid assay formats for the detection of reporter enzymes that are widely used in gene expression studies. Chemiluminescent detection methodologies typically provide up to 100-1000x higher sensitivities than can be achieved with the corresponding fluorescent or colorimetric enzyme substrates. The varieties of 1,2-dioxetane substrates available provides assay versatility, allowing optimization of assay formats with the available instrumentation, and are ideal for use in gene expression assays performed in both biomedical and pharmaceutical research. These assays are amenable to automation with a broad range of instrumentation for high throughput compound screening.

Alkaline Phosphatase↗
PubMed2000

Quantitative polymerase chain reaction and solid-phase capture nucleic acid detection.

The combination of PCR amplification and chemiluminescent detection of PCR products provides a highly sensitive system for the quantitation of DNA and RNA. The broad dynamic range of the chemiluminescent detection assay simplifies the selection of cycling and concentration parameters critical to harnessing the quantitative aspects of PCR amplification. Detection of 200 amol of PCR product is attained using the described procedures. The tube or microplate format of the assay avoids many of the limitations associated with other methods of PCR quantitation involving gel electrophoresis. This detection methodology can be applied to a variety of quantitative nucleic acid assays, including viral load and gene expression analysis.

DNA↗
PubMed2000

Novel methods for chemiluminescent detection of reporter enzymes.

Chemiluminescent reporter gene assays provide highly sensitive, quantitative detection in simple, rapid assay formats for detection of reporter enzymes that are widely employed in gene expression studies. Chemiluminescent detection methodologies typically provide up to 100-1000x higher sensitivities than may be achieved with fluorescent or colorimetric enzyme substrates. The variety of chemiluminescent 1,2-dioxetane substrates available enable assay versatility, allowing optimization of assay formats with the available instrumentation, and are ideal for use in gene expression assays performed in both biomedical and pharmaceutical research. In addition, 1,2,-dioxetane chemistries can be multiplexed with luciferase detection reagents for dual detection of multiple enzymes in a single sample. These assays are amenable to automation with a broad range of instrumentation for high throughput compound screening.

3T3 Cells↗
PubMed1998

Metastasis-associated Mts1 (S100A4) protein modulates protein kinase C phosphorylation of the heavy chain of nonmuscle myosin.

Mts1 protein (S100A4 according to a new classification) has been implicated in the formation of the metastatic phenotype via regulation of cell motility and invasiveness. Previously we have demonstrated that Mts1 protein interacted with the heavy chain of nonmuscle myosin in a calcium-dependent manner. To elucidate the role of the Mts1-myosin interaction, we mapped the Mts1-binding region on the myosin heavy chain molecule. We prepared proteolytically digested platelet myosin and a series of overlapped myosin heavy chain protein fragments and used them in a blot overlay with Mts1 protein. Here we report that the Mts1-binding site is located within a 29-amino acid region, at the C-terminal end of the myosin heavy chain (between 1909-1937 amino acids). Two-dimensional phosphopeptide analysis showed that Mts1 protein inhibits protein kinase C phosphorylation of the platelet myosin heavy chain at Ser-1917. We hypothesize that Mts1 protein regulates cytoskeletal dynamics of the metastatic cells through modulation of the myosin phosphorylation by protein kinase C in calcium-dependent fashion.

Binding Sites↗
PubMed1996

Comparison of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione and luminol as co-substrates for detection of horseradish peroxidase in enhanced chemiluminescent reactions.

The utility of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione (HDP) as a co-substrate for the chemiluminescent detection of horseradish peroxidase was assessed. Several substituted aryl boronic acid derivatives (4-phenyl, 4-iodo) acted as potent enhancers of the peroxidase catalyzed reaction. Addition of chelating agents (EDTA) and surfactants (Tween-20 and [poly (vinylbenzyl)tributylphosphonium chloride-poly (vinylbenzyl) trioctylphosphonium chloride copolymer]) modulated background light emission and the intensity and duration of the signal from both HDP and luminol. However, HDP was found to be inferior to luminol in the peroxidase assay. Comparative studies revealed that at 500 amol of peroxidase the S/B was ten-fold higher using a commercial luminol-based signal reagent as compared with an HDP-EDTA-Tween-20 reagent (S/B t = 0 min 21.8 vs 1.7, S/B t = 10 min 17.8 vs 2.0).

Boronic Acids↗
PubMed1996

Dual luminescence-based reporter gene assay for luciferase and beta-galactosidase.

A unique combined luminescence assay for firefly (Photinus pyralis) luciferase and beta-galactosidase (beta-gal) reporter gene products is described. Luciferase and beta-gal activities are determined with the same aliquot of cell lysate prepared from cells contransfected with both reporter genes, thereby reducing manual labor and increasing experimental accuracy. With the Dual-Light assay system, luciferase activity is measured first with an enhanced luciferase assay, followed by quantitation of beta-gal with Galacton-Plus chemiluminescent substrate and Sapphire-II enhancer. Highly sensitive detection of luciferase (2 fg) and beta-gal (8 fg) is achieved with a dynamic range over seven orders of magnitude of enzyme concentration. Comparative analysis of both independent and combined (Dual-Light) detection methods for cells contransfected with luciferase and beta-gal reporter genes is also described.

Animals↗
PubMed1996

Chemiluminescence: sensitive detection technology for reporter gene assays.

A series of enzyme-activated chemiluminescence-based assays of reporter gene expression, useful in many biomedical applications, has been developed. The chemiluminescence detection systems for beta-galactosidase, beta-glucuronidase (GUS), and secreted placental alkaline phosphatase (SEAP) reporter enzymes are all based on use of 1,2-dioxetane substrates. This detection technology also permits the combined luminescence detection of two different reporter enzymes in the same tube, e.g., a dual assay for beta-galactosidase and luciferase. The sensitivity of these chemiluminescence assays is several orders of magnitude greater than that of conventional colorimetric or fluorometric detection methods; e.g., the detection limit for beta-galactosidase by the chemiluminescence assay is 8 fg and by a fluorometric assay is 2 pg. Furthermore, chemiluminescence enables detection of beta-galactosidase, GUS, and SEAP enzyme concentrations over a dynamic range of more than five to six orders in magnitude. These assays offer highly sensitive, quantitative, rapid, nonisotopic detection of reporter enzymes that are widely used in both mammalian cells and plant cells.

Alkaline Phosphatase↗
PubMed1995

Quantitation of PCR products with chemiluminescence.

Quantitative PCR and reverse transcription PCR (RT-PCR) are widely used in biomedical, industrial and other research applications to determine the number of RNA or DNA molecules of a specific type and/or sequence in a sample of interest. We have developed an assay system to accurately quantitate PCR products that utilizes solid-phase capture and an enzyme-linked chemiluminescent detection method. The entire assay is performed in a single tube or microplate well. Biotinylated PCR products are quantitated by capture onto a streptavidin-coated surface, followed by hybridization of an internal fluorescein-labeled oligonucleotide probe and subsequent detection with an anti-fluorescein-alkaline phosphatase conjugate and CSPD chemiluminescent substrate. Light signal is measured in a luminometer. The assay sensitivity enables accurate quantitation of target DNA because the measurement is performed on product generated during the exponential phase of amplification. The broad dynamic range of the assay, which is greater than three orders of magnitude of PCR product concentration, simplifies the determination of the number of amplification cycles necessary for accurate quantitation of target molecules. The PCR-Light system is an ultrasensitive, non-isotopic and rapid assay for PCR product detection that also has general application to solution hybridization assays and other quantitation methods.

Alkaline Phosphatase↗
PubMed1994

Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products.

Chemiluminescent assays are described for the secreted alkaline phosphatase (SEAP) and beta-glucuronidase (GUS) reporter gene products. These assays provide simple, sensitive, non-isotopic alternatives to existing detection methods and are performed in microplate or tube luminometers or in a scintillation counter. The SEAP reporter gene product is secreted from mammalian cells and is thus easily detected in a sample of culture medium. Sensitive detection of secreted placental alkaline phosphatase is performed with CSPD chemiluminescent alkaline phosphatase substrate, and approximately 3 fg of enzyme can be detected. GUS has become the major reporter gene used for the analysis of plant gene expression. Sensitive chemiluminescent detection of GUS activity can be performed with an assay system we have developed using Glucuron, a beta-glucuronidase substrate. This chemiluminescent assay detects 60 fg of GUS and is linear over six orders of magnitude of enzyme concentration. CSPD and Glucuron substrates have been incorporated into two new chemiluminescent reporter gene assay kits for SEAP and GUS.

Alkaline Phosphatase↗