Combinatorial microscopy.

By taking advantage of combinations of the many rich properties of photons, new forms of optical microscopy can now be used to visualize features of samples beyond thickness and density variations. We are now within reach of viewing the motions, orientations, binding kinetics and specific transient associations of previously 'submicroscopic' cellular structures and single molecules.

Effects of polychlorinated biphenyls, hexachlorocyclohexanes, and mercury on human neutrophil apoptosis, actin cytoskelton, and oxidative state.

Apoptosis, or programmed cell death, has been proposed as a biomarker for environmental contaminant effects. In this work, we test the hypothesis that in vitro assays of apoptosis are sensitive indicators of immunological effects of polychlorinated biphenyls, hexachlorocyclohexanes, and mercury on human neutrophils. Apoptosis, necrosis, and viability as well as the related indicators F-actin levels, and active thiol state were measured in purified human neutrophils after treatment with contaminants.

Multiple receptor states are required to describe both kinetic binding and activation of neutrophils via N-formyl peptide receptor ligands.

It is well-established that the binding of N-formyl peptides to the N-formyl peptide receptor on neutrophils can be described by a kinetic scheme that involves two ligand-bound receptor states, both a low affinity ligand-receptor complex and a high affinity ligand-receptor complex, and that the rate constants describing ligand-receptor binding and receptor affinity state interconversion are ligand-specific. Here we examine whether differences due to these rate constants, i.e.

Effects of selected polybrominated diphenyl ether flame retardants on lake trout (Salvelinus namaycush).

Polybrominated diphenyl ether (PBDE) flame-retardants have been identified as an emergent contaminants issue in many parts of the world. In vitro analyses were conducted to test the hypothesis that selected PBDEs congeners affect viability, apoptosis, and necrosis of thymocytes from laboratory-reared lake trout (Salvelinus namaycush). At current environmental levels (< 1 mg/L), effects of the tested PBDEs on thymocytes were negligible.

G protein threshold behavior in the human neutrophil oxidant response: measurement of G proteins available for signaling in responding and nonresponding subpopulations.

Threshold behavior is an important aspect of signal transduction pathways that allows for responses to be turned on or off. Human neutrophil responses to N-formyl peptides, including oxidant production and release, exhibit threshold behavior with respect to the number of G proteins available for signaling; progressive treatment of neutrophils with pertussis toxin causes the conversion of responding cells to nonresponding cells. To quantify the threshold level of G proteins required for signaling of N-formyl peptide stimulated oxidant production in a neutrophil population, we used a plasma membrane associated G protein quantification assay in conjunction with a sorting flow cytometer and measured differences in the average number of G proteins available for signaling per cell in both the responding and the nonresponding subpopulations after pertussis toxin treatment.

Lindane stimulates neutrophils by selectively activating phospholipase C and phosphoinositide-kinase activity.

The organochlorine insecticide lindane is a known activator of neutrophil responses. In this work we delineated the biochemical pathways by which lindane stimulates neutrophil oxidant production. Plasma membrane GTPase activity was not stimulated by lindane, ruling out a role for lindane-induced activation of G-proteins or G-protein coupled receptors, whereas inhibition of phospholipase C inhibited lindane-induced oxidant production.

Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.

The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state.

Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins.

A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol.

Priming-induced localization of G(ialpha2) in high density membrane microdomains.

Subcellular fractionation of human neutrophils on linear sucrose density gradients was utilized to test the hypothesis that priming regulates the subcellular and sub-plasma membrane distribution of neutrophil G-protein subunits, G(ialpha2) and G(ialpha3), N-formyl peptide receptor, Lyn kinase and phospholipase C(beta2). G(ialpha2), but not G(ialpha3), moved from a lighter to a higher density plasma membrane fraction. Unoccupied N-formyl peptide receptors were found throughout the plasma membrane fractions and this distribution did not change with priming.

In vitro toxicity and interactions of environmental contaminants (Arochlor 1254 and mercury) and immunomodulatory agents (lipopolysaccharide and cortisol) on thymocytes from lake trout (Salvelinus namaycush).

The immunotoxicity of chemical combinations commonly encountered by the lake trout (Salvelinus namaycush) immune system was the focus of this study. It was hypothesised that combinations of an environmental contaminant (mercuric chloride or Aroclor 1254) and an immunomodulatory agent (bacterial endotoxin or cortisol) might interact to produce a greater toxicity than that of the environmental contaminant alone at concentrations typically encountered in piscine blood and other tissues. Thus lake trout thymocytes were isolated and treated with mercuric chloride or Aroclor 1254 in the presence and absence of cortisol or lipopolysaccharide.

Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands.

Background: Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor-ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand.

Inhibition of mono-ADP-ribosyltransferase activity during the execution phase of apoptosis prevents apoptotic body formation.

The objective of this study was to understand factors responsible for apoptotic body formation and release during apoptosis. We have found that inhibition of mono-ADP ribosylation after ultraviolet (UV) light induction of apoptosis in HL-60 cells does not block caspase-3 activation, gelsolin cleavage, or endonucleolytic DNA fragmentation. However, the cytoskeletal features of apoptosis leading to apoptotic body formation and release were inhibited by meta-iodobenzylguanidine (MIBG) and novobiocin, potent inhibitors of arginine-specific mono-ADP-ribosyltransferases (mono-ADPRTs).

Actin cytoskeletal function is spared, but apoptosis is increased, in WAS patient hematopoietic cells.

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS.

Modeling activation and desensitization of G-protein coupled receptors provides insight into ligand efficacy.

Signaling through G-protein coupled receptors is one of the most prevalent and important methods of transmitting information to the inside of cells. Many mathematical models have been proposed to describe this type of signal transduction, and the ternary complex (ligand/receptor/G-protein) model and its derivatives are among the most widely accepted. Current versions of these equilibrium models include both active (i.

Regulation of oscillations in filamentous actin content in polymorphonuclear leukocytes stimulated with leukotriene B(4) and platelet-activating factor.

Stimulation of neutrophils with LTB(4) or PAF results in the production of a rapidly oscillating actin polymerization/depolymerization response. Treatment of neutrophils with inhibitors of PKC prior to stimulation with ligand resulted in a masking of the F-actin oscillations. Because myosin has been shown to be a substrate for neutrophil PKC, this protein was investigated as a potential downstream mediator of F-actin oscillations.

Granulocyte-macrophage colony-stimulating factor rescues human polymorphonuclear leukocytes from ultraviolet irradiation-accelerated apoptosis.

Introduction: Bacterial lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) delay PMN apoptosis during in vitro culture. The present study was undertaken to determine if LPS and GM-CSF can rescue UV-irradiated PMN from undergoing apoptosis and to determine the role of extracellular signal-regulated kinase (ERK) in this process.

Materials And Methods: PMN were preincubated with LPS (20 ng/ml) and GM-CSF (100 units/ml) for 60 min before being UV-irradiated for 15 min.

A mathematical model for ligand/receptor/G-protein dynamics and actin polymerization in human neutrophils.

A mathematical model is proposed for describing the dynamics of the chemotactic peptide-stimulated actin polymerization response in human neutrophils. The response pathway utilizes the guanine nucleotide binding protein (G-protein) signal transduction cascade common to many receptor systems and allows adaptation in the continued presence of ligand. The development of such a model is an important first step toward understanding, predicting, and ultimately manipulating neutrophil responses.

Threshold and graded response behavior in human neutrophils: effect of varying G-protein or ligand concentrations.

Observing the qualitative characteristics of response behavior as key variables in the signal transduction cascade are changed can provide insight into the fundamental roles of these interactions in producing cellular responses. Using flow cytometric assays and pertussis toxin (PT) treatment of human neutrophils, we have shown that actin polymerization stimulated with the chemoattractants N-formyl-Met-Leu-Phe, leukotriene B4, and interleukin-8 exhibits threshold behavior in terms of G-protein number. Partial PT treatment resulted in both responding and nonresponding populations of cells upon stimulation.

Experimental approaches for observing homologous desensitisation and their pitfalls.

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses have been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. These are (1) observation of attenuation of a response after an initial stimulus-induced activation, (2) repeated stimulation of cells after washing away the previous stimulus, and (3) repeated stimulation without a wash step.

Experimental approaches for observing homologous desensitisation and their pitfalls.

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses has been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. These are (1) observation of attenuation of a response after an initial stimulus-induced activation, (2) repeated stimulation of cells after washing away the previous stimulus, and (3) repeated stimulation without a wash step.

Lipopolysaccharide protects polymorphonuclear leukocytes from apoptosis via tyrosine phosphorylation-dependent signal transduction pathways.

Background: The present study was undertaken to determine if tyrosine phosphorylation signal transduction pathways, which are known to be activated in polymorphonuclear leukocytes (PMN) by lipopolysaccharide (LPS), play a role in priming of PMN oxidative burst and protection of PMN from apoptosis by LPS, and to determine if an interface between these two signaling pathways exists.

Methods: PMN were combined with or without 10-fold serial dilutions (0.1 ng-1 microgram/ml) of LPS and incubated at 37 degrees C/5% CO2.

Autocrine/paracrine modulation of polymorphonuclear leukocyte survival after exposure to Candida albicans.

Polymorphonuclear leukocytes (PMN) play a central role in the host response to injury and infection. These terminally differentiated phagocytes have a limited life span, after which they undergo spontaneous apoptosis. PMN life span can be significantly prolonged by several naturally occurring cytokines, and PMN are now known to be capable of cytokine production in response to various antigenic stimuli.

Calculation of diffusion-limited kinetics for the reactions in collision coupling and receptor cross-linking.

Both enzyme (e.g., G-protein) activation via a collision coupling model and the formation of cross-linked receptors by a multivalent ligand involve reactions between two molecules diffusing in the plasma membrane.

Ultraviolet irradiation accelerates apoptosis in human polymorphonuclear leukocytes: protection by LPS and GM-CSF.

Polymorphonuclear leukocytes (PMN) play a central role in host response to injury and infection. Understanding factors that regulate PMN survival may therefore have a major influence on the development of novel treatment strategies for controlling life-threatening infections, as well as local and systemic inflammatory responses. Unfortunately, the presently utilized in vitro culture model of PMN apoptosis makes the examination of early biochemical events surrounding PMN apoptosis very difficult.

Membrane-proximal calcium transients in stimulated neutrophils detected by total internal reflection fluorescence.

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within approximately 100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe.