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Peter O Krutzik

Publications and source records attributed to Peter O Krutzik.

12 recordsLinked to original sources
PubMed2006

Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence.

We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of beta-galactosidase (beta-gal) activity. The substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered cells and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of beta-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant beta-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of beta-gal permits bioluminescent imaging applications that previously were not possible.

Animals↗
PubMed2006

Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.

Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.

Animals↗
PubMed2005

Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry.

Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.

Animals↗
PubMed2005

Characterization of the murine immunological signaling network with phosphospecific flow cytometry.

The immune system is a multitiered network that at the first level uses changes to intracellular signaling proteins to commit cells to determined fates. At the second tier, cells interact with one another via specifically expressed surface receptors and their cognate signaling molecules. At the third level, the local environments of immune cells change the outcomes of intracellular signaling pathways and thereby the role of cells during immune challenge. The interplay among these three tiers allows the distinct cell types of the immune system to respond cohesively to eliminate foreign Ags. In this study, using phosphospecific flow cytometry, we analyze elements of these network tiers by generating profiles of single-cell phosphoprotein responses in B cells, T cells, and myeloid cells to a number of mechanistically and clinically relevant cytokines (IFN-gamma, GM-CSF, IL-2, and IL-10) as well as LPS at key regulatory interfaces (Jak-Stat and MAPK pathways). The stimuli typically induced phosphorylation of specific signaling pathways and exerted their effects on distinct subsets of immune cells. However, upon comparison of stimulation in vitro and in vivo, we noted that signaling pathway specificity and cell type specificity were influenced strongly by the external environment. When taken from the in vivo environment, certain cell subsets became hypo- or hyper-responsive, showed profound differences in sensitivity to cytokine levels, or displayed altered phosphorylation kinetics. Thus, simultaneous analysis of the three tiers of the immune system network illustrates the principles by which immune regulation is context dependent and how in vitro culture systems compare with the in vivo environment.

Animals↗
PubMed2004

Single cell profiling of potentiated phospho-protein networks in cancer cells.

Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cancer cell survival and pathology. Profiles of cancer cell signaling networks might therefore identify mechanisms by which such cells interpret environmental cues for continued growth. Using multiparameter flow cytometry, we monitored phospho-protein responses to environmental cues in acute myeloid leukemia at the single cell level. By exposing cancer cell signaling networks to potentiating inputs, rather than relying upon the basal levels of protein phosphorylation alone, we could discern unique cancer network profiles that correlated with genetics and disease outcome. Strikingly, individual cancers manifested multiple cell subsets with unique network profiles, reflecting cancer heterogeneity at the level of signaling response. The results revealed a dramatic remodeling of signaling networks in cancer cells. Thus, single cell measurements of phospho-protein responses reveal shifts in signaling potential of a phospho-protein network, allowing for categorizing of cell network phenotypes by multidimensional molecular profiles of signaling.

Antineoplastic Agents↗
PubMed2004

Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications.

Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phospho-epitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins.

Animals↗
PubMed2004

Flow cytometric analysis of kinase signaling cascades.

Flow cytometry offers the capability to assess the heterogeneity of cellular subsets that exist in complex populations, such as peripheral blood, based on immunophenotypes. We describe methodologies to measure phospho-epitopes in single cells as determinants of intracellular kinase activity. Multiparametric staining, using both surface and intracellular stains, allows for the study of discrete biochemical events in readily discernible lymphocyte subsets. As such, the usage of multiparameter flow cytometry to obtain proteomic information provides several major advantages: (1) the ability to perform multiparametric experiments to identify distinct signaling profiles in defined lymphocyte populations, (2) simultaneous correlation of multiple active kinases involved in signaling cascades, (3) profiling of active kinase states to identify signaling signatures of interest rapidly, and (4) biochemical access to rare cell subsets such as those from clinically derived samples or populations that comprise too few in numbers for conventional biochemical analysis.

Animals↗
PubMed2003

Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events.

BACKGROUND: Recent advances in intracellular staining techniques, cytometer technology, fluorescent reagents, and antibody production have expanded the number of intracellular antigens that can be analyzed by flow cytometry. Measurement of protein phosphorylation with phospho-specific antibodies has given insight into kinase signaling cascades. However, available techniques for phospho-epitope staining can differ greatly, making it necessary to understand the differences between the outcomes when such techniques are applied and to develop robust and reproducible methods of application. METHODS: Ten different cellular fixation and permeabilization techniques were tested for their ability to provide phospho-specific staining. Combinations of formaldehyde, methanol, ethanol, acetone, Triton X-100, and saponin were used as fixation and permeabilization reagents. Phospho-specific antibodies were labeled with Alexa Fluor dyes to provide multicolor analysis of different signaling events simultaneously within individual cells. RESULTS: Fixing cells with 1.5% formaldehyde followed by permeabilization in methanol gave optimal results for pERK, pp38, pJNK, pStat1, pStat5, and pStat6 staining. Alteration of formaldehyde fixation and methanol permeabilization times affected measurements of phosphorylation induction. Phospho-specific flow cytometric analyses correlated well with Western blotting, providing cross platform validation of the technique. CONCLUSIONS: Measuring phosphorylation events by flow cytometry provides a rapid and efficient way to measure kinase cascades in individual cells. Stability of phospho-epitopes in methanol allows long-term storage of samples prior to analysis. Multiple signaling cascades can be monitored simultaneously through the use of different fluorophore labels to determine specificity of ligands or inhibitors. Application of optimized techniques to heterogeneous cell types such as peripheral blood or murine splenocytes may allow signaling to be analyzed simultaneously in immune cell subsets.

Antibodies, Monoclonal↗
PubMed2002

Rapid solid-phase synthesis of DNA-binding pyrrole-imidazole polyamides.

Pyrrole-imidazole polyamides can be synthesized to target predetermined sequences of DNA with nanomolar affinity and high specificity, and have been shown to modulate gene transcription both in vitro and in vivo. To make polyamides more readily available to biological laboratories, we have developed a rapid solid-phase synthesis based on azabenzotriazole (OAt) activation that decreases synthesis time 60% compared to standard benzotriazole (OBt) techniques, without loss of yield or purity.

DNA↗
PubMed2002

5'-alkyl-benzothiadiazides: a new subgroup of AMPA receptor modulators with improved affinity.

AMPA receptors form a major subdivision of the glutamate receptor family that mediates excitatory synaptic transmission in the brain. Currents through AMPA receptors can be up- or down-regulated by compounds that allosterically modulate receptor kinetics through binding sites distinct from that for glutamate. One of those modulators is the benzothiadiazide IDRA-21 which has been reported to enhance synaptic transmission and be effective in behavioral tests, but typically requires threshold concentrations of at least 100 microM to be active in vitro. In this study, new benzothiadiazides were developed with IDRA-21 as lead compound and examined for their potency in modulating AMPA receptor kinetics. A significant increase in drug affinity was obtained by alkyl substitution at the 5'-position of IDRA-21; substitutions at other positions of the benzothiadiazide core generally did not yield a further gain in affinity and in some cases abolished drug binding. The 5'-ethyl derivative exhibited an EC(50) value in the order of 22 microM which represents about a 30-fold gain in affinity over that of IDRA-21. The EC(50) value is comparable to that of cyclothiazide, the most potent benzothiadiazide drug, but the effects on AMPA receptors differed substantially between these two compounds in that the 5'-ethyl derivative of IDRA-21 greatly increased the binding affinity for receptor agonists whereas cyclothiazide is known to reduce agonist binding. The structure--activity relationships reported here thus offer to provide new insights how receptor kinetics is linked to particular aspects of receptor--drug interactions.

Animals↗