Desorption/ionization on silicon nanowires.

Dense arrays of single-crystal silicon nanowires (SiNWs) have been used as a platform for laser desorption/ionization mass spectrometry of small molecules, peptides, protein digests, and endogenous and xenobiotic metabolites in biofluids. Sensitivity down to the attomole level has been achieved on the nanowire surfaces by optimizing laser energy, surface chemistry, nanowire diameter, length, and growth orientation. An interesting feature of the nanowire surface is that it requires lower laser energy as compared to porous silicon and MALDI to desorb/ionize small molecules, therefore reducing background ion interference. Taking advantage of their high surface area and fluid wicking capabilities, SiNWs were used to perform chromatographic separation followed by mass analysis of the separated molecules providing a unique platform that can integrate separation and mass spectrometric detection on a single surface.

A role for p21-activated kinase in endothelial cell migration.

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and increased stress fibers and focal adhesions. The DN effect mapped to the most NH(2)-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged alpha-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.

p21-activated protein kinase: a crucial component of morphological signaling?

The mechanisms by which Rho family GTPases (Rho, Rac and Cdc42) regulate coordinated changes to the actin cytoskeleton are being elucidated. This review will focus on the current evidence that the p21-activated kinases (PAKs) are involved in regulating some of the diverse cytoskeletal changes induced by Rac and Cdc42. PAKs have been shown to be required for processes including neurite formation and axonal guidance, development of cell polarity and motile responses. Signaling molecules interacting with PAKs that might contribute to the regulation of such processes have recently been identified.

alphaPix stimulates p21-activated kinase activity through exchange factor-dependent and -independent mechanisms.

Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind alphaPix. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 was also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.

A GTPase-independent mechanism of p21-activated kinase activation. Regulation by sphingosine and other biologically active lipids.

p21-activated kinases (PAKs) are serine/threonine kinases that have been identified as targets for the small GTPases Rac and Cdc42. PAKs have been implicated in cytoskeletal regulation, stimulation of mitogen-activated protein kinase cascades, and in control of the phagocyte NADPH oxidase. Membrane targeting of PAK1 induced increased kinase activity in a GTPase-independent manner, suggesting that other mechanisms for PAK regulation exist. We observed concentration- and time-dependent activation of PAK1 by sphingosine and several related long chain sphingoid bases but not by ceramides or a variety of other lipids. Although phospholipids were generally ineffective, phosphatidic acid and phosphatidylinositol also had stimulatory effects on PAK1. Lipid stimulation induced a similar level of PAK1 activity as did stimulation by GTPases, and the patterns of PAK1 autophosphorylation determined after partial tryptic digestion and two-dimensional peptide analysis were similar with each class of activator. Lipid stimulation of PAK1 activity was dependent upon intact PAK kinase activity, as indicated by studies with a kinase-dead PAK1 mutant. Treatment of COS-7 cells expressing wild type PAK1 with sphingosine, fumonisin B, or sphingomyelinase, all of which are able to elevate the levels of free sphingosine, induced increased activity of PAK1 as determined using a p47(phox) peptide substrate. Studies using PAK1 mutants suggest that lipids act at a site overlapping or identical to the GTPase-binding domain on PAK. The inactive sphingosine derivative N,N-dimethylsphingosine was an effective inhibitor of PAK1 activation in response to either sphingosine or Cdc42. Our results demonstrate a novel GTPase-independent mechanism of PAK activation and, additionally, suggest that PAK(s) may be important mediators of the biological effects of sphingolipids.

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.

Localization of p21-activated kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in stimulated cells.

The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics. We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src-transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.

Endothelial cell-associated platelet-activating factor primes neutrophils for enhanced superoxide production and arachidonic acid release during adhesion to but not transmigration across IL-1 beta-treated endothelial monolayers.

The role of platelet-activating factor (PAF) in stimulating neutrophils during interaction with HUVEC has been investigated using the specific PAF receptor antagonists WEB 2086 and YM 264. PAF antagonists at concentrations up to 40 microM had little effect on the adhesion of neutrophils to control or IL-1 beta-stimulated HUVEC monolayers. However, polarization of neutrophils on adhesion to IL-1-treated HUVEC was markedly diminished in the presence of PAF antagonists. In addition, the PAF antagonists WEB 2086 and YM 264 strongly inhibited the migration of neutrophils across IL-1-treated endothelial monolayers. Priming of neutrophil respiratory burst and arachidonic acid release caused by contact with IL-1-treated endothelial cells was also inhibited by PAF antagonists. However, priming as a consequence of transmigration was not inhibited by either WEB 2086 or YM 264. These results suggest that HUVEC-associated PAF plays a central role in the polarization and subsequent transmigration of neutrophils during interaction with HUVEC monolayers. Moreover, the results further suggest that PAF is responsible for priming neutrophils during contact with HUVECs. However, after transmigration, factors other than PAF are responsible for the priming of neutrophil function.

Priming of the oxidative burst in human neutrophils by physiological agonists or cytochalasin B results from the recruitment of previously non-responsive cells.

Using a sensitive flow cytometric assay, which measures the intracellular oxidation of 2'7' dichlorofluorescein (DCFH) by H2O2, we have assessed, at a single-cell level, the effects of a variety of physiological priming agonists and cytochalasin B (CB) on purified populations of neutrophils stimulated at different points along the signal response transduction pathway. Pretreatment of purified neutrophils with the physiological priming agonists monocyte interleukin-8 (IL-8), granulocyte-monocyte colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, and non-stimulatory doses of formyl-methionyl-leucyl-phenylalanine (FMLP), resulted in an increased percentage of cells generating an oxidative burst in response to subsequent receptor stimulation with FMLP. CB had a similar but much more pronounced effect on cellular recruitment to a receptor-mediated responsive state. Activation of protein kinase C (PKC) using the phorbol ester phorbol myristate acetate (PMA) resulted in a heterogeneous response, with all cells generating H2O2, but with two populations differing in their magnitude of response. Physiological priming agonists had no effect on the heterogeneity of the PMA response. However, pretreatment with CB dramatically altered the PMA response, producing a homogeneous population highly responsive to stimulation with PKC. In contrast, direct stimulation of G proteins with fluoride (A1F-4) was primed both by physiological priming agonists and by CB. These results demonstrate that priming of neutrophils by physiological agonists involves changes at the level of signal transduction which enable a previously non-responsive cell to respond to a secondary stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-stimulated Cl- efflux from human neutrophils. A common phenomenon during neutrophil activation.

When human peripheral blood neutrophils were stimulated with various agonists which activate and/or prime neutrophils, we found that Cl- efflux was enhanced with a dramatic (50%) loss of intracellular Cl-. Interestingly, the Cl- efflux was enhanced by both agonists which induce a rapid transient increase in intracellular Ca2+ concentration ([Ca2+]i) [class I, e.g. N-formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL8), platelet-activating factor, leukotriene B4 and C5a] and those which do not induce such an [Ca2+]i elevation [class II, e.g. tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. The time course of agonist-stimulated Cl- efflux differed depending on the agonist. Class I agonists such as IL8 and fMLP exhibited a 1 min lag phase before the onset of Cl- efflux; class II agonists such as GM-CSF and TNF displayed a 2 and 5 min lag phase, respectively. Both IL8 (class I)- and TNF (class II)-stimulated Cl- efflux exhibited similar sensitivity to inhibition by different types of ion transport inhibitors [ethacrynic acid (EA), amiloride, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, anthracene-9-carboxylic acid, and 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid]. On the other hand, natural Cl- efflux, which is thought to be mainly mediated by Cl-/Cl- self exchange, was not inhibited by EA (0.5 mM) or amiloride (0.3 mM). These results imply that both class I and class II agonist-stimulated Cl- efflux occurs via a common Cl- transporter which is different from that reported previously in resting human neutrophils. Although all agonists which induced a Cl- efflux also induced shape change of neutrophils, there did not appear to be a causal relationship between shape change and agonist-stimulated Cl- efflux. However, a temporal correlation was found to exist between agonist-stimulated Cl- efflux and intracellular alkalinization following agonist stimulation. Agonist-stimulated Cl- efflux therefore seems to be a common phenomenon activated by several agonists which act through different signal transduction pathways.

Differential regulation of early phase and late phase responses in human neutrophils by cAMP.

The elevation of intracellular levels of cyclic AMP by forskolin stimulation of adenylate cyclase regulates early and late phase neutrophil responses differentially. Early phase neutrophil responses as measured by shape change in response to chemotactic factors, transmigration across a polycarbonate membrane and priming were unaffected by forskolin-induced elevation of intracellular cAMP. Late phase neutrophil responses such as release of superoxide anions, activation of phospholipase A2 and platelet activating factor (PAF) synthesis were inhibited by increasing intracellular cAMP through the addition of 10 microM forskolin for 10 min prior to stimulation. N-Formyl-methionyl-leucyl-phenylalanine-stimulated arachidonic acid release fell from 9.3% (untreated cells) to 4.6% in forskolin-treated cells. PAF generation was also inhibited from 430 pg/10(6) cells in untreated cells to background levels in forskolin-treated cells (110 pg/10(6) cells). Also, the reduction of cytochrome c by superoxide anions fell from 4.2 nmol/10(6) cells in the absence of forskolin to 2.0 nmol/10(6) cells following forskolin treatment. These results indicate that in neutrophils the elevation of cAMP acts differentially on cellular responses, not affecting early activation events, but markedly inhibiting late events such as the release of inflammatory mediators.

Recombinant human monocyte IL-8 primes NADPH-oxidase and phospholipase A2 activation in human neutrophils.

We demonstrate for the first time that recombinant human monocyte interleukin-8 (rhMIL-8) primes human neutrophil responses to fMLP. Human neutrophils preincubated for 10 min with 10(-8) M rhMIL-8 and then stimulated with micromolar fMLP show enhanced release of superoxide anions, platelet-activating factor (PAF) and arachidonic acid compared with cells which are not initially exposed to rhMIL-8. We also demonstrate that this enhancement of the neutrophil response is dependent on the dose of rhMIL-8 with the greatest enhancement corresponding with IL-8 levels which cause maximum shape change of neutrophils. Priming of neutrophils occurred after only 30 seconds preincubation with rhMIL-8 indicating that the mechanism of IL-8 priming is extremely rapid as was stimulation of neutrophil shape change by rhMIL-8. Priming of neutrophils with rhMIL-8 did not increase sensitivity to fMLP but enhanced responsiveness to activating concentrations. rhMIL-8 alone at levels used for priming caused no release of superoxide anions, arachidonic acid or PAF. These results suggest that IL-8 primes neutrophil phospholipase A2 and NADPH-oxidase activation in response to fMLP.

Terminal complement complexes and C1/C1 inhibitor complexes in rheumatoid arthritis and other arthritic conditions.

Terminal complement complex (TCC) and C1r-C1s-C1 inhibitor complex (C1/C1 INH) concentrations were measured in plasma and synovial fluid from patients with arthritis and related to other measures of disease activity. Both TCC and C1/C1 INH concentrations were significantly increased in patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis (plasma and synovial fluid, P less than 0.05) and normal subjects (plasma only, P less than 0.001). In the patients with RA, there was no correlation between plasma or synovial fluid TCC concentrations and IgM rheumatoid factor, immune complex or C1/C1 INH levels. However, in 10 patients with seronegative RA, C1/C1 INH and immune complex levels correlated significantly in synovial fluid (r = 0.69, P less than 0.05) although not in plasma (r = 0.52). Plasma and synovial fluid TCC and C1/C1 INH concentrations did not differ in rheumatoid patients with severe compared with mild joint disease (categorized by the Ritchie score). These results confirm a role for complement activation in RA but suggest that several mechanisms are involved in its pathogenesis.

Human rheumatoid synovial cell stimulation by the membrane attack complex and other pore-forming toxins in vitro: the role of calcium in cell activation.

The effects of non-lethal amounts of a variety of pore-forming agents on cultured human rheumatoid synovial cells (HRSC) have been investigated. Non-lethal complement membrane attack and non-lethal amounts of melittin, perforin and ionomycin all caused a biphasic release of prostaglandin E2 (PGE2) from HRSC, an early phase of release occurring within 1 hr and a second larger phase commencing after 4 hr and continuing over the 24-hr time-course. Removal of extracellular calcium abolished the release of PGE2 under all conditions of non-lethal attack. Modulation of G-protein activity reduced the second phase of release caused by non-lethal doses of the membrane-attack complex (MAC) from 800 ng/10(6) cells PGE2 to around 300 ng/10(6) cells. Non-lethal levels of the MAC also caused release of interleukin-6 (IL-6) from HRSC over the 24-hr time-course, with levels reaching 550 ng/10(6) cells at 24 hr compared to background levels of 200 ng/10(6) cells. No detectable release of IL-1 alpha could be measured at any time following non-lethal complement membrane attack. These results suggest a role for the MAC as an initiating mediator inducing the inflammation associated with rheumatoid arthritis.

Human protectin (CD59), an 18,000-20,000 MW complement lysis restricting factor, inhibits C5b-8 catalysed insertion of C9 into lipid bilayers.

Human cells are relatively resistant to lysis by the homologous complement system. Here we describe the mechanism of action of a recently discovered and widely distributed 18,000-20,000 molecular weight (MW) membrane glycoprotein (CD59), which appears to act as a major protective element against complement-mediated lysis (hence called protectin). When incorporated into heterologous erythrocyte membranes, protectin efficiently prevented cell lysis by human serum. Neutralization with antibody of the naturally occurring protectin on human erythrocytes or on nucleated K562 cells increased their susceptibility to lysis by homologous complement. During complement activation, protectin became incorporated into the membrane attack complex (MAC). By interacting with newly exposed regions in the C5b-8 complex and in aggregating C9 it limited the number of C9 molecules associating with the C5b-8 complex to a C8:C9 ratio of 1:1.5 instead of a normal average of 1:3.5. The results demonstrate directly that protectin is a powerful inhibitor of complement cytolysis and acts by inhibiting the C5b-8 catalysed insertion of C9 into the lipid bilayer.

Stimulation of human rheumatoid synovial cells by non-lethal complement membrane attack.

The effects of non-lethal complement attack on cultured human rheumatoid synovial cells have been investigated by measuring a variety of parameters. Within 3-4 min of initiating non-lethal complement membrane attack there was a rise in reactive oxygen metabolite release from cultured synovial cells, which slowly returned to basal levels over a period of 45 min. The response was dependent on the formation of the complete C5b-9 complex. Prostaglandin E2 was also released during non-lethal attack in a biphasic manner, an early phase of release occurring within the first hour and a second, larger phase commencing at 4 hr and rising to levels of over 1000 ng/10(6) cells at 24 hr, compared to control levels at this time of less than 100 ng/10(6) cells. This response was dependent on the formation of the C5b-8 complex but did not require C9. Removal of extracellular calcium reduced release of prostaglandin E2 to background levels, and inclusion of an inhibitor of protein synthesis abolished the second phase of release but not the first phase. Non-lethal attack caused release of small amounts of leukotriene B4 but no detectable release of tumour necrosis factor.

In vivo and in vitro evidence of cell recovery from complement attack in rheumatoid synovium.

In the previous article we have demonstrated, by quantifying terminal complement complexes in synovial fluid, that membrane attack complex activation occurs in the joint in rheumatoid arthritis. Here we describe evidence of synoviocyte resistance to complement attack in vivo and in vitro. Gel filtration of terminal complement complex positive synovial fluid on Sepharose 2B revealed two forms of terminal complement complex: one form, eluting coincident with the column void, did not react with antibody to the fluid-phase inhibitor of complement membrane attack, the S-protein, suggesting that it was composed of membrane attack complexes, the other form, eluting in the included volume, did react with the anti-S-protein antibody, suggesting that it was composed of functionally inactive SC5b-9 complexes. The high molecular weight membrane attack complex peak was demonstrated by electron microscopy to be composed of membrane vesicles bearing many lesions having the typical appearance of complement membrane attack complexes. No discernible structures were present in the lower molecular weight peak. The effects of non-lethal complement membrane attack on human synoviocytes in culture were also investigated. Synoviocytes were relatively resistant to killing by autologous complement, end-point lysis of optimally antibody-sensitized cells never exceeding 60% even at a serum dilution of 1:2. At serum dilutions of 1:20 or less, no significant cell killing occurred despite a high degree of membrane attack pathway activation, suggesting the existence of resistance and recovery mechanisms. Non-lethal complement membrane attack stimulated the release of toxic reactive oxygen metabolites from synoviocytes. These, and other reactive species released during non-lethal complement attack in vivo, may play a significant role in the pathogenesis of rheumatoid arthritis.