Jan Andries Post

C11-BODIPY581/591, an oxidation-sensitive fluorescent lipid peroxidation probe: (Micro)spectroscopic characterization and validation of methodology

C11-BODIPY(581/591) is a fluorescent radio-probe for indexing lipid peroxidation and antioxidant efficacy in model membrane systems and living cells, with excellent characteristics: (i) emission in the visible range of the electromagnetic spectrum, with good spectral separation of the nonoxidized (595 nm) and oxidized (520 nm) forms; (ii) has a high quantum yield and because of this, low labeling concentrations can be used, ensuring minimal perturbation of the membrane whilst retaining favorable signal to noise ratios; (iii) has a good photo-stability and displays very few fluorescence artifacts; (iv) is virtually insensitive to environmental changes, i.e., pH or solvent polarity; (v) is lipophilic and as such easily enters membranes; (vi) once oxidized, C11-BODIPY(581/591) remains lipophilic and does not spontaneously leave the lipid bilayer; (vii) C11-BODIPY(581/591) localizes in two distinct pools within the lipid bilayer, a shallow pool at 18 A and a deep pool at < 7.5 A from the center of the bilayer; (viii) is not cytotoxic to rat-1 fibroblasts up to 50 microM; (ix) is sensitive to a variety of oxy-radicals and peroxynitrite, but not to superoxide, nitric oxide, transition metal ions, and hydroperoxides per se; (x) its sensitivity to oxidation is comparable to that of endogenous fatty acyl moieties.

RATIO-FLUORESCENCE MICROSCOPY OF LIPID OXIDATION IN LIVING CELLS USING C11-BODIPY581/591

A ratio‐fluorescence assay was developed for on‐line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11‐BODIPY581/591. Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide‐induced oxidation of C11‐BODIPY581/591 was visualized at the sub‐cellular level in rat‐1 fibroblasts. Pre‐loading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11‐BODIPY581/591 is a valuable tool to quantify lipid oxidation and anti‐oxidant efficacy in single cells.

Tetrahydrobiopterin, but Not l-Arginine, Decreases NO Synthase Uncoupling in Cells Expressing High Levels of Endothelial NO Synthase

Endothelial NO synthase (eNOS) produces superoxide when depleted of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) and l-arginine by uncoupling the electron flow from NO production. High expression of eNOS has been reported to have beneficial effects in atherosclerotic arteries after relatively short periods of time. However, sustained high expression of eNOS may have disadvantageous vascular effects because of uncoupling. We investigated NO and reactive oxygen species (ROS) production in a microvascular endothelial cell line (bEnd.3) with sustained high eNOS expression and absent inducible NOS and neuronal NOS expression using 4,5-diaminofluorescein diacetate and diacetyldichlorofluorescein as probes, respectively. Unstimulated cells produced both NO and ROS. After stimulation with vascular endothelial growth factor (VEGF), NO and ROS production increased. VEGF-induced ROS production was even further increased by the addition of extra l-arginine. N&ohgr;-nitro-l-arginine methyl ester decreased ROS production. These findings strongly suggest that eNOS is a source of ROS in these cells. Although BH4 levels were increased as compared with another endothelial cell line, eNOS levels were >2 orders of magnitude higher. The addition of BH4 resulted in increased NO production and decreased generation of ROS, indicating that bEnd.3 cells produce ROS through eNOS uncoupling because of relative BH4 deficiency. Nevertheless, eNOS-dependent ROS production was not completely abolished by the addition of BH4, suggesting intrinsic superoxide production by eNOS. This study indicates that potentially beneficial sustained increases in eNOS expression and activity could lead to eNOS uncoupling and superoxide production as a consequence. Therefore, sustained increases of eNOS or VEGF activity should be accompanied by concomitant supplementation of BH4.

Cellular solid-state nuclear magnetic resonance spectroscopy

Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution.

Impact of agglomeration state of nano- and submicron sized gold particles on pulmonary inflammation

BackgroundNanoparticle (NP) toxicity testing comes with many challenges. Characterization of the test substance is of crucial importance and in the case of NPs, agglomeration/aggregation state in physiological media needs to be considered. In this study, we have addressed the effect of agglomerated versus single particle suspensions of nano- and submicron sized gold on the inflammatory response in the lung. Rats were exposed to a single dose of 1.6 mg/kg body weight (bw) of spherical gold particles with geometric diameters of 50 nm or 250 nm diluted either by ultrapure water or by adding phosphate buffered saline (PBS). A single dose of 1.6 mg/kg bw DQ12 quartz was used as a positive control for pulmonary inflammation. Extensive characterization of the particle suspensions has been performed by determining the zetapotential, pH, gold concentration and particle size distribution. Primary particle size and particle purity has been verified using transmission electron microscopy (TEM) techniques. Pulmonary inflammation (total cell number, differential cell count and pro-inflammatory cytokines), cell damage (total protein and albumin) and cytotoxicity (alkaline phosphatase and lactate dehydrogenase) were determined in bronchoalveolar lavage fluid (BALF) and acute systemic effects in blood (total cell number, differential cell counts, fibrinogen and C-reactive protein) 3 and 24 hours post exposure. Uptake of gold particles in alveolar macrophages has been determined by TEM.ResultsParticles diluted in ultrapure water are well dispersed, while agglomerates are formed when diluting in PBS. The particle size of the 50 nm particles was confirmed, while the 250 nm particles appear to be 200 nm using tracking analysis and 210 nm using TEM. No major differences in pulmonary and systemic toxicity markers were observed after instillation of agglomerated versus single gold particles of different sizes. Both agglomerated as well as single nanoparticles were taken up by macrophages.ConclusionPrimary particle size, gold concentration and particle purity are important features to check, since these characteristics may deviate from the manufacturers description. Suspensions of well dispersed 50 nm and 250 nm particles as well as their agglomerates produced very mild pulmonary inflammation at the same mass based dose. We conclude that single 50 nm gold particles do not pose a greater acute hazard than their agglomerates or slightly larger gold particles when using pulmonary inflammation as a marker for toxicity.

Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C

In this study we show that phosphorylation of extracellular signal‐regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO−) exposure occurs via a MAPK kinase (MEK)‐independent but PKC‐dependent pathway in rat‐1 fibroblasts. ONOO−‐mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO− treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down‐regulation of PKC by phorbol‐12,13‐dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO−, implying that activation of ERK by ONOO− depends on activation of PKC. Indeed, PKCα and ϵ were activated upon ONOO− exposure. When cells were treated with ONOO− in a calcium‐free buffer, no activation of PKCα was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCα and ERK phosphorylation by ONOO−. Indeed, ONOO− exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using Gö6976, an inhibitor of calcium‐dependent PKC activation, implied that ONOO−‐mediated ERK1/2 phosphorylation depends on activation of a calcium‐dependent PKC.

The influence of reactive oxygen species on cell cycle progression in mammalian cells.

Cell cycle regulation is performed by cyclins and cyclin dependent kinases (CDKs). Recently, it has become clear that reactive oxygen species (ROS) influence the presence and activity of these enzymes and thereby control cell cycle progression. In this review, we first describe the discovery of enzymes specialized in ROS production: the NADPH oxidase (NOX) complexes. This discovery led to the recognition of ROS as essential players in many cellular processes, including cell cycle progression. ROS influence cell cycle progression in a context-dependent manner via phosphorylation and ubiquitination of CDKs and cell cycle regulatory molecules. We show that ROS often regulate ubiquitination via intermediate phosphorylation and that phosphorylation is thus the major regulatory mechanism influenced by ROS. In addition, ROS have recently been shown to be able to activate growth factor receptors. We will illustrate the diverse roles of ROS as mediators in cell cycle regulation by incorporating phosphorylation, ubiquitination and receptor activation in a model of cell cycle regulation involving EGF-receptor activation. We conclude that ROS can no longer be ignored when studying cell cycle progression.

The Porosity, Acidity, and Reactivity of Dealuminated Zeolite ZSM-5 at the Single Particle Level: The Influence of the Zeolite Architecture

A combination of atomic force microscopy (AFM), high-resolution scanning electron microscopy (HR-SEM), focused-ion-beam scanning electron microscopy (FIB-SEM), X-ray photoelectron spectroscopy (XPS), confocal fluorescence microscopy (CFM), and UV/Vis and synchrotron-based IR microspectroscopy was used to investigate the dealumination processes of zeolite ZSM-5 at the individual crystal level. It was shown that steaming has a significant impact on the porosity, acidity, and reactivity of the zeolite materials. The catalytic performance, tested by the styrene oligomerization and methanol-to-olefin reactions, led to the conclusion that mild steaming conditions resulted in greatly enhanced acidity and reactivity of dealuminated zeolite ZSM-5. Interestingly, only residual surface mesoporosity was generated in the mildly steamed ZSM-5 zeolite, leading to rapid crystal coloration and coking upon catalytic testing and indicating an enhanced deactivation of the zeolites. In contrast, harsh steaming conditions generated 5-50 nm mesopores, extensively improving the accessibility of the zeolites. However, severe dealumination decreased the strength of the Brønsted acid sites, causing a depletion of the overall acidity, which resulted in a major drop in catalytic activity.

Membrane Phospholipid Asymmetry and Signal Transduction

It is now 75 years ago that the lipid bilayer concept was proposed by Gorter and Grendel (1925). Since then many concepts about the organization of lipids and proteins in the membrane have been proposed, including the milestone of the “fluid mosaic bilayer concept” of Singer and Nicolson (1972). In this concept (glyco)proteins can laterally diffuse within the fluid (glyco)lipid bilayer. This concept is appealing in its simplicity, but it is now generally accepted that there can be many domains of proteins within the fluid bilayer. These include, interaction with cytoskeletal proteins, such as coated vesicles, or in cellular functionalities such as gap junctions and desmosomes, or in the well-known crystalline domain of the purple membrane or under conditions where lipid phase transitions by solidification of lipids introduce protein domains in the membrane ( see for review Verkleij, 1989). In the recent years it has been suggested that in rafts and/or caveolae, which are enriched in sphingomyelin, cholesterol and glycolipid, the lipids induce domains in which specific proteins are present (Pralle et al., 2000). These proteins fit in these domains because they match with theira-helices within the thickness of the lipid bilayer in these domains (for reviews seeKillian, 1998; Dumas et al., 1999). Besides domains within the plane of the membrane, differences in the two membrane halves exist, the socalled lipid asymmetry. This asymmetry and its role in cellular (dys)functioning and signal transduction is the topic of this review.

Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide.

The membrane-permeable fluorescein-labelled tyramine conjugate (acetylTyrFluo) was used to identify the proteins of normal human dermal fibroblasts most susceptible to oxidation by hydrogen peroxide [Van der Vlies, Wirtz and Pap (2001) Biochemistry 40, 7783-7788]. By exposing the cells to H(2)O(2) (0.1 mM for 10 min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [(35)S]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the (35)S-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the (35)S-labelled proteins and identified by comparison with our mastermap of proteins. Protein disulphide isomerase (PDI), IgG-binding protein (BiP), calnexin, endoplasmin and glucose-regulated protein 58 (endoplasmic reticulum protein 57/GRP58) were identified as targets of oxidation. All these proteins reside in the endoplasmic reticulum and are part of the protein folding machinery. In agreement, confocal laser scanning microscopy showed co-localization of TyrFluo-labelled proteins and the KDEL receptor ERD-2, a marker for the endoplasmic reticulum.