Stochastic methods for aerosol chemistry: a compact molecular description of functionalization and fragmentation in the heterogeneous oxidation of squalane aerosol by OH radicals.

The heterogeneous oxidation of organic aerosol by hydroxyl radicals (OH) can proceed through two general pathways: functionalization, in which oxygen functional groups are added to the carbon skeleton, and fragmentation, in which carbon-carbon bonds are broken, producing higher volatility, lower molecular weight products. An ongoing challenge is to develop a quantitative molecular description of these pathways that connects the oxidative evolution of the average aerosol properties (e.g. size and hygroscopicity) to the transformation of free radical intermediates. In order to investigate the underlying molecular mechanism of aerosol oxidation, a relatively compact kinetics model is developed for the heterogeneous oxidation of squalane particles by OH using free radical intermediates that convert reactive hydrogen sites into oxygen functional groups. Stochastic simulation techniques are used to compare calculated system properties over ten oxidation lifetimes with the same properties measured in experiment. The time-dependent average squalane aerosol mass, volume, density, carbon number distribution of scission products, and the average elemental composition are predicted using known rate coefficients. For functionalization, the calculations reveal that the distribution of alcohol and carbonyl groups is controlled primarily by the initial OH abstraction rate and to lesser extent by the branching ratio between secondary peroxy radical product channels. For fragmentation, the calculations reveal that the formation of activated alkoxy radicals with neighboring functional groups controls the molecular decomposition, particularly at high O/C ratios. This kinetic scheme provides a framework for understanding the oxidation chemistry of a model organic aerosol and informs parameterizations of more complex systems.

Oxidation of a model alkane aerosol by OH radical: the emergent nature of reactive uptake.

An accurate description of the evolution of organic aerosol in the Earth's atmosphere is essential for climate models. However, the complexity of multiphase chemical and physical transformations has been challenging to describe at the level required to predict aerosol lifetimes and changes in chemical composition. In this work a model is presented that reproduces experimental data for the early stages of oxidative aging of squalane aerosol by hydroxyl radical (OH), a process governed by reactive uptake of gas phase species onto the particle surface. Simulations coupling free radical reactions and Fickian diffusion are used to elucidate how the measured uptake coefficient reflects the elementary steps of sticking of OH to the aerosol as a result of a gas-surface collision, followed by very rapid abstraction of hydrogen and subsequent free radical reactions. It is found that the uptake coefficient is not equivalent to a sticking coefficient or an accommodation coefficient: it is an intrinsically emergent process that depends upon particle size, viscosity, and OH concentration. An expression is derived to examine how these factors control reactive uptake over a broad range of atmospheric and laboratory conditions, and is shown to be consistent with simulation results. Well-mixed, liquid behavior is found to depend on the reaction conditions in addition to the nature of the organic species in the aerosol particle.

Method of measuring the spatial resolution of a photoresist.

By analysis of the response of a high-contrast photoresist to sinusoidal illumination, generated interferometrically, one can extract a phenomenological modulation transfer function of the resist material, thereby characterizing its spatial resolution. Deep-ultraviolet interferometric lithography allows the resist response to be quantified at length scales below 100 nm. As an example, the resolution (FWHM) of the commercial resist UVII-HS is found to be approximately 50 nm. This simple method can be applied to materials under development for advanced photolithography with short-wavelength illumination.

Transendothelial migration of colon carcinoma cells requires expression of E-selectin by endothelial cells and activation of stress-activated protein kinase-2 (SAPK2/p38) in the tumor cells.

Adhesion and migration of tumor cells on and through the vascular endothelium are critical steps of the metastatic invasion. We investigated the roles of E-selectin and of stress-activated protein kinase-2 (SAPK2/p38) in modulating endothelial adhesion and transendothelial migration of HT-29 colon carcinoma cells. Tumor necrosis factor alpha (TNF alpha) strongly increased the expression of E-selectin in human umbilical vein endothelial cells (HUVEC). This effect was independent of the activation of SAPK2/p38 induced by TNF alpha. Adhesion of HT-29 cells on a monolayer of HUVEC pretreated with TNF alpha was dependent on E-selectin expression but was independent of SAPK2/p38 activity of both HUVEC and tumor cells. The adhesion of HT-29 cells to E-selectin-expressing HUVEC led to the activation of SAPK2/p38 in the tumor cells as reflected by the increased phosphorylation of the actin-polymerizing factor HSP27 by mitogen-activated protein kinase 2/3, a direct target of SAPK2/p38. Moreover, a recombinant E-selectin/Fc chimera quickly increased the activation of SAPK2/p38 in HT-29 cells. Blocking the increased activity of SAPK2/p38 of HT-29 cells by SB203580 or by expressing a dominant negative form of SAPK2/p38 inhibited their transendothelial migration. Similarly, HeLa cells stably expressing a kinase-inactive mutant of SAPK2/p38 showed a decreased capacity to cross a layer of HUVEC. Overall, our results suggest that the regulation of transendothelial migration of tumor cells involves two essential steps as follows: adhesion to the endothelium through adhesion molecules, such as E-selectin, and increased motogenic potential through adhesion-mediated activation of the SAPK2/p38 pathway.

p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle.

Angiotensin II (ANG II) is a multifunctional hormone that exerts potent vasoconstrictor and hypertrophic effects on vascular smooth muscle. Here, we demonstrate that the p38 mitogen-activated protein (MAP) kinase pathway is involved in ANG II-induced vascular contraction. Addition of ANG II to rat aortic smooth muscle cells (SMC) caused a rapid and transient increase of p38 activity through activation of the AT(1) receptor subtype. This response to ANG II was strongly attenuated by pretreating cells with antioxidants and diphenylene iodonium and was mimicked by exposure of cells to H(2)O(2). Stimulation of p38 by ANG II resulted in the enzymatic activation of MAP kinase-activated protein (MAPKAP) kinase-2 and the phosphorylation of heat shock protein 27 (HSP27) in aortic SMC. Pretreatment of cells with the specific p38 MAP kinase inhibitor SB-203580 completely blocked the ANG II-dependent activation of MAPKAP kinase-2 and phosphorylation of HSP27. ANG II also caused a robust activation of MAPKAP kinase-2 in the intact rat aorta. Incubation with SB-203580 significantly decreased the potency of ANG II to induce contraction of rat aortic rings and depressed the maximal hormone response. These results suggest that the p38 MAP kinase pathway selectively modulates the vasoconstrictor action of ANG II in vascular smooth muscle.

Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase.

In endothelial cells, vascular endothelial growth factor (VEGF) induces an accumulation of stress fibers associated with new actin polymerization and rapid formation of focal adhesions at the ventral surface of the cells. This cytoskeletal reorganization results in an intense motogenic activity. Using porcine endothelial cells expressing one or the other type of the VEGF receptors, VEGFR1 or VEGFR2, or human umbilical vein endothelial cells pretreated with a VEGFR2 neutralizing antibody, we show that VEGFR2 is responsible for VEGF-induced activation of the stress-activated protein kinase-2/p38 (SAPK2/p38), phosphorylation of focal adhesion kinase (FAK), and enhanced migratory activity. Activation of SAPK2/p38 triggered actin polymerization whereas FAK, which was phosphorylated independently of SAPK2/p38, initiated assembly of focal adhesions. Both processes contributed to the formation of stress fibers. Geldanamycin, an inhibitor of HSP90 blocked tyrosine phosphorylation of FAK, assembly of focal adhesions, actin reorganization, and cell migration, all of which were reversed by overexpressing HSP90. We conclude that VEGFR2 mediates the physiological effect of VEGF on cell migration and that two independent pathways downstream of VEGFR2 regulate actin-based motility. One pathway involves SAPK2/p38 and leads to enhanced actin polymerization activity. The other involves HSP90 as a permissive signal transduction factor implicated in FAK phosphorylation and assembly of focal adhesions.

Integrating the VEGF signals leading to actin-based motility in vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor that stimulates endothelial cell proliferation and migration, two key events of the angiogenic process. The intracellular signals leading to these events have recently been investigated and a better understanding on how VEGF induces its angiogenic functions is emerging. Herein, we summarize recent findings on how VEGF stimulates endothelial cell migration and contributes to angiogenesis. In particular, the role of the VEGF receptors involved in initiating the coordinated signals that leads to actin-based motility is discussed.

SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis.

In endothelial cells, H2O2 induces the rapid formation of focal adhesion complexes at the ventral face of the cells and a major reorganization of the actin cytoskeleton into dense transcytoplasmic stress fibers. This change in actin dynamics results from the activation of the mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38), which, via MAP kinase-activated protein (MAPKAP) kinase-2/3, leads to the phosphorylation of the actin polymerization modulator heat shock protein of 27 kD (HSP27). Here we show that the concomitant activation of the extracellular signal-regulated kinase (ERK) MAP kinase pathway by H2O2 accomplishes an essential survival function during this process. When the activation of ERK was blocked with PD098059, the focal adhesion complexes formed under the plasma membrane, and the actin polymerization activity led to a rapid and intense membrane blebbing. The blebs were delimited by a thin F-actin ring and contained enhanced levels of HSP27. Later, the cells displayed hallmarks of apoptosis, such as DEVD protease activities and internucleosomal DNA fragmentation. Bleb formation but not apoptosis was blocked by extremely low concentrations of the actin polymerization inhibitor cytochalasin D or by the SAPK2 inhibitor SB203580, indicating that the two processes are not in the same linear cascade. The role of HSP27 in mediating membrane blebbing was assessed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosphorylatable HSP27, H2O2 did not induce F-actin accumulation, nor did it generate membrane blebbing activity in the presence or absence of PD098059. In contrast, in fibroblasts that expressed wild-type HSP27 to a level similar to that found in endothelial cells, H2O2 induced accumulation of F-actin and caused bleb formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little ERK activity, also induced membrane blebbing that was dependent on the expression of HSP27. In these cells, membrane blebbing was not followed by caspase activation or DNA fragmentation. We conclude that the HSP27-dependent actin polymerization-generating activity of SAPK2 associated with a misassembly of the focal adhesions is responsible for induction of membrane blebbing by stressing agents.

Role of the mitogen-activated protein kinases in the expression of the kinin B1 receptors induced by tissue injury.

Several cytokines and LPS regulate the population of the B1 receptors (B1Rs) for kinins; these are responsive to des-Arg9-bradykinin (BK) and Lys-des-Arg9-BK. B1R activation contributes to inflammatory vascular changes and pain. Aortic rings isolated from normal rabbits and incubated in vitro in Krebs physiological medium were used as a model of tissue injury. From a null level of response, these rings exhibit a time- and protein synthesis-dependent increase in the maximal contractile response to des-Arg9-BK. Exposure to exogenous IL-1beta or epidermal growth factor (EGF) considerably increases the process of sensitization to the kinins. Freshly isolated control aortic rings showed high mitogen-activated protein (MAP) kinase activities (persistent activation of p38, but less prolonged for extracellular signal-regulated kinase and c-Jun-N-terminal kinase/stress-activated protein kinase pathways) relatively to the basal activities found in various types of cultured cells. IL-1beta or EGF further increased the activities of the extracellular signal-regulated kinase and c-Jun-N-terminal kinase/stress-activated protein kinase MAP kinases. The inhibitor of the p38 MAP kinase, SB 203580 (10 microM), massively (approximately 75%) and selectively inhibited the spontaneous sensitization to des-Arg9-BK over 6 h. SB 203580 also significantly reduced the development of the response to des-Arg9-BK as stimulated by IL-1 or EGF. Both spontaneous and IL-1beta-stimulated up-regulation of responsiveness to des-Arg9-BK were significantly inhibited by the MAP kinase extracellular signal-regulated kinase kinase 1 inhibitor PD 98059 (approximately 40%). The protein kinase inhibitors failed to inhibit protein synthesis and to acutely inhibit the contractile effect of des-Arg9-BK, suggesting that they do not influence B1 receptor transduction mechanisms. In cultured aortic smooth muscle cells stimulated with EGF, MAP kinase activation preceded B1R mRNA induction. Protein kinase inhibitors reveal the role of cell injury-controlled MAP kinase pathways, and singularly of the p38 pathway, in the induction of B1R.

p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent chemotactic agent for endothelial cells. Yet the signalling pathways that modulate the motogenic effects of VEGF in vascular endothelial cells are still ill defined. In the present study, we found in primary cultures of human umbilical vein endothelial cells (HUVEC) that VEGF increased cell migration and induced a marked reorganization of the microfilament network that was characterized by the formation of stress fibers and the recruitment of vinculin to focal adhesions. VEGF also stimulated the mitogen activated protein (MAP) kinases ERK (extracellular signal-regulated kinase) and p38 (stress activated protein kinase-2), but not SAPK1/JNK (stress activated protein kinase-1/c-Jun NH2-terminal kinase). Activation of p38 resulted in activation of MAP kinase activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). Inhibiting the VEGF-induced activation of ERK with PD098059 did not influence actin organization or cell migration but totally inhibited the VEGF-induced incorporation of thymidine into DNA. Inhibition of p38 activity by the specific inhibitor SB203580 led to an inhibition of HSP27 phosphorylation, actin reorganization and cell migration. The results indicate that the p38 pathway conveys the VEGF signal to microfilaments inducing rearrangements of the actin cytoskeleton that regulate cell migration. By modulating cell migration, p38 may thus be an important regulator of angiogenesis.

Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells.

Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H2O2 in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles, H2O2 also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H2O2-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.

HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress.

Phosphorylation of heat shock protein 27 (HSP27) has been suggested to play an important role in the regulation of F-actin dynamics in response to growth factors and stress. Because the microfilament network is one of the earliest targets of oxidative stress and because phosphorylation of HSP27 is strongly induced by reactive oxygen metabolites, we have investigated the role of HSP27 phosphorylation in regulating actin dynamics in response to oxidative stress. Experiments were done in Chinese hamster CCL39 cells overexpressing various levels of the wild-type or a nonphosphorylatable form of human HSP27 (pm3 HSP27). In control cells, both H2O2 and menadione induced fragmentation of F-actin, which forms aggregates and patches concentrated around the nucleus. Stable overexpression of wild-type HSP27, but not of pm3 HSP27, conferred resistance against actin fragmentation, suggesting that HSP27 has a phosphorylation-activated protective function against actin disruption by oxidative stress. Cell lines that overexpressed the highest levels of the wild-type form of human HSP27 also showed an increased cell survival following exposure to H2O2. In contrast, cells expressing pm3 HSP27 were as sensitive as the controls to the lethal effect of H2O2. These results suggest that phosphorylation of HSP27 is causally related to the regulation of microfilament dynamics following oxidative stress and may be involved in mediating an adaptive response to oxyradical-generating agents such as carcinogens, anticancer drugs, and other xenobiotics.

Characterization of 45-kDa/54-kDa HSP27 kinase, a stress-sensitive kinase which may activate the phosphorylation-dependent protective function of mammalian 27-kDa heat-shock protein HSP27.

Heat-shock protein 27 (HSP27) is a major target of phosphorylation upon cell stimulation with a variety of agents and has been suggested to have a phosphorylation-regulated function at the level of actin filaments. Here we investigated comparatively the mechanisms of HSP27 phosphorylation by oxidative stresses, exposures to tumor necrosis factor (TNF), heat shock and growth factors. Extracts of Chinese hamster or human cells exposed to H2O2, xanthine/xanthine oxidase, menadione or TNF contained up to 15-fold more HSP27 kinase activity than comparable extracts obtained from control cells. Induction of HSP27 kinase activity by TNF or H2O2 was completely inhibited by first treating the cells with the antioxidant N-acetyl-L-cysteine, suggesting that generation of reactive oxygen metabolites was the key triggering element of this induction. In contrast, prior treatment with acetylcysteine had no or little effect on the induction by thrombin, serum and heat shock. The kinase activity in extracts of cells stimulated by heat shock, H2O2, sodium arsenite, TNF or growth factors was identified by in-gel renaturation and purified approximately 8000-fold by sequential chromatography. In all cases, the induced kinase activity was entirely associated with two polypeptides of 45 kDa and 54 kDa, identified as mitogen-activated-protein kinase-activated protein (MAPKAP) kinase-2 based on its reactivation in vitro by 42/44-kDa MAP kinases, its antigenic properties and its substrate specificity. The 45/54-kDa HSP27 kinase may play an important role in the cell response to oxidative stress. Overexpression of the wild-type HSP27 but not of a nonphosphorylatable form of human HSP27 in Chinese hamster cells conferred resistance to actin fragmentation by oxidative stress generated by H2O2. It is concluded that activation of the 45/54-kDa HSP27 kinase is a common mechanism of HSP27 phosphorylation to which converge both oxyradical-dependent and oxyradical-independent pathways and which may participate in a homeostatic response to stress at the level of actin microfilament.