Structure and dynamics of the human pleckstrin DEP domain: distinct molecular features of a novel DEP domain subfamily

Pleckstrin1 is a major substrate for protein kinase C in platelets and leukocytes, and comprises a central DEP (disheveled, Egl-10, pleckstrin) domain, which is flanked by two PH (pleckstrin homology) domains. DEP domains display a unique alpha/beta fold and have been implicated in membrane binding utilizing different mechanisms. Using multiple sequence alignments and phylogenetic tree reconstructions, we find that 6 subfamilies of the DEP domain exist, of which pleckstrin represents a novel and distinct subfamily. To clarify structural determinants of the DEP fold and to gain further insight into the role of the DEP domain, we determined the three-dimensional structure of the pleckstrin DEP domain using heteronuclear NMR spectroscopy. Pleckstrin DEP shares main structural features with the DEP domains of disheveled and Epac, which belong to different DEP subfamilies. However, the pleckstrin DEP fold is distinct from these structures and contains an additional, short helix alpha4 inserted in the beta4-beta5 loop that exhibits increased backbone mobility as judged by NMR relaxation measurements. Based on sequence conservation, the helix alpha4 may also be present in the DEP domains of regulator of G-protein signaling (RGS) proteins, which are members of the same DEP subfamily. In pleckstrin, the DEP domain is surrounded by two PH domains. Structural analysis and charge complementarity suggest that the DEP domain may interact with the N-terminal PH domain in pleckstrin. Phosphorylation of the PH-DEP linker, which is required for pleckstrin function, could regulate such an intramolecular interaction. This suggests a role of the pleckstrin DEP domain in intramolecular domain interactions, which is distinct from the functions of other DEP domain subfamilies found so far.     

Isolation and characterisation of the major outer membrane protein of Erwinia carotovora

Treatment of the tonoplast H(+)-ATPase from mung bean seedlings (Vigna radiata L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was calculated to be 0.98, suggesting that at least one histidine residue of vacuolar H(+)-ATPase was modified by DEP. The absorbance of the vacuolar H(+)-ATPase at 240 nm was progressively increased after incubation with DEP, suggesting that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine, reversed the absorbance change and partially restored the enzymic activity. The pK(a) of modified residues of vacuolar H(+)-ATPase was kinetically determined to be 6.73, a value close to that of histidine. Thus, it is assuredly concluded that histidine residues of the vacuolar H(+)-ATPase were modified by DEP. Kinetic analysis showed that V(max) but not K(m) of vacuolar H(+)-ATPase was decreased by DEP. This result is interpreted as that the residual activity after DEP inhibition was primarily due to the unmodified enzyme molecules. Moreover, simultaneous presence of DEP and DCCD (N,N'-dicyclohexyl-carbodiimide), an inhibitor modified at proteolipid subunit of vacuolar H(+)-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further demonstrate that only one out of four histidine residues modified was involved in the inhibition of vacuolar H(+)-ATPase by DEP. Mg(2+)-ATP, the physiological substrate of vacuolar H(+)-ATPase, but not its analogs, exerted preferentially partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate.     

Rosmarinic acid inhibits lung injury induced by diesel exhaust particles

Epidemiological and experimental studies have suggested that diesel exhaust particles (DEP) may be involved in recent increases in lung diseases. DEP has been shown to generate reactive oxygen species. Intratracheal instillation of DEP induces lung inflammation and edema in mice. Rosmarinic acid is a naturally occurring polyphenol with antioxidative and anti-inflammatory activities. We investigated the effects of rosmarinic acid on lung injury induced by intratracheal administration of DEP (500 microg/body) in mice. Oral supplementation with administration of rosmarinic acid (2 mg/body for 3 d) inhibited DEP-induced lung injury, which was characterized by neutrophil sequestration and interstitial edema. DEP enhanced the lung expression of keratinocyte chemoattractant (KC), interleukin-1beta, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1alpha, which was inhibited by treatment with rosmarinic acid. DEP enhanced expression of iNOS mRNA and formation of nitrotyrosine and 8-OHdG in the lung, which was also inhibited by rosmarinic acid. These results suggest that rosmarinic acid inhibits DEP-induced lung injury by the reduction of proinflammatory molecule expression. Antioxidative activities of rosmarinic acid may also contribute to its protective effects.     

Isolation and quantitative estimation of diesel exhaust and carbon black particles ingested by lung epithelial cells and alveolar macrophages in vitro

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After various incubation periods, the cells were separated from free extracellular DEP or CB particles by Ficoll density gradient centrifugation and dissolved in hot sodium dodecyl sulfate detergent. Insoluble DEP or CB residues were isolated by high-speed centrifugation, and the elemental carbon (EC) concentrations in the pellets were estimated by a thermal-optical-transmittance method (i.e., carbon analysis). From the EC concentration, the amount of ingested DEP or CB could be calculated. The described technique allowed the determination of the kinetics and dose dependence of DEP uptake by LA4 lung epithelial cells and MHS alveolar macrophages. Both cell types ingested DEP to a similar degree; however, the MHS macrophages took up significantly more CB than the epithelial cells. Cytochalasin D, an agent that blocks actin polymerization in the cells, inhibited approximately 80% of DEP uptake by both cell types, indicating that the process was actin-dependent in a manner similar to phagocytosis. This technique can be applied to examine the interactions between cells and particles containing EC and to study the modulation of particle uptake in diseased tissue.     

Pulmonary exposure to diesel exhaust particles enhances coagulatory disturbance with endothelial damage and systemic inflammation related to lung inflammation

Pulmonary exposure to diesel exhaust particles (DEP) enhances lung inflammation related to bacterial endotoxin (lipopolysaccharide [LPS]) in mice. Severe lung inflammation can reportedly induce coagulatory abnormalities and systemic inflammation. This study examined the effects of components of DEP on lung inflammation, pulmonary permeability, coagulatory changes, systemic inflammatory response, and lung-to-systemic translocation of LPS in a murine model of lung inflammation. ICR mice were divided into six experimental groups that intratracheally received vehicle, LPS (2.5 mg/kg), organic chemicals in DEP (DEP-OC; 4 mg/kg) extracted with dicloromethane), residual carbonaceous nuclei of DEP (washed DEP: 4 mg/kg), DEP-OC + LPS, or washed DEP + LPS. Both DEP components exacerbated lung inflammation, vascular permeability, and the increased fibrinogen and E-selectin levels induced by LPS. With overall trends, the exacerbation was more prominent with washed DEP than with DEP-OC. Washed DEP + LPS significantly decreased activated protein C and antithrombin-III and elevated circulatory levels of interleukin (IL)-6, keratinocyte chemoattractant (KC), and LPS as compared with LPS alone, whereas DEP-OC + LPS elevated IL-6, KC, and LPS without significance. These results show that DEP components, especially washed DEP, amplify the effects if LPS on the respiratory system and suggest that they contribute to the adverse health effects of particulate air pollution on the sensitive populations with predisposing vascular and/or pulmonary diseases, including ischemic vascular diseases and respiratory infection.     

Induction of systemic Th1 and Th2 immune responses by oral administration of soluble antigen and diesel exhaust particles

The present study was undertaken to examine whether oral administration of soluble antigen together with diesel exhaust particles (DEP) induced the systemic immune response in mice. Mice were orally given 1 mg of hen egg lysozyme (HEL) with varying doses of DEP every 3 days over a period of 15 days. The results showed that oral administration of HEL plus DEP produced anti-HEL IgG antibodies in serum in a dose-related fashion, while either HEL or DEP alone failed to show the antigen-specific IgG antibody production. Production of anti-HEL IgG2a and IgG1 antibodies, which are dependent on Th1 and Th2 CD4(+) T cells, respectively, was seen in mice fed with combined HEL and DEP, although anti-HEL IgG1 antibodies appeared to be more efficiently produced by lower doses of DEP than anti-HEL IgG2a antibodies. There was marked antigen-specific proliferation of spleen cells in mice treated with HEL and DEP. The anti-HEL antibody production and lymphoid cell proliferation to the antigen were associated with marked secretion of the Th1 cytokine IFN-gamma as well as the Th2 cytokine IL-4. These results suggest that DEP may act as a mucosal adjuvant in the gut enhancing systemic Th1 and Th2 immune responses and might play a role in oral immunization and food allergy.     

Thioredoxin-1 suppresses lung injury and apoptosis induced by diesel exhaust particles (DEP) by scavenging reactive oxygen species and by inhibiting DEP-induced downregulation of Akt

Diesel exhaust particles (DEP) are reactive oxygen species (ROS)-inducing toxic agents that damage lungs. Thioredoxin-1 (Trx-1) is a thiol protein with antioxidant and redox-regulating effects. In this study, we demonstrate that Trx-1 scavenges ROS generated by DEP and attenuates the lung injury. Intratracheal instillation of DEP resulted in the generation of more hydroxyl radicals in control mice than in human Trx-1 (hTrx-1)-transgenic mice as measured by noninvasive L-band in vivo electron spin resonance. DEP caused acute lung damage with massive infiltration of inflammatory cells in control mice, but much less damage in hTrx-1-transgenic mice. The hTrx-1 transgene protected the mice against DEP toxicity. To investigate further the molecular mechanism of the protective role of Trx-1 against DEP-induced lung injury, we used hTrx-1-transfected L-929 cells and recombinant hTrx-1 (rhTrx-1)-pretreated A-549 cells. DEP-induced ROS generation was suppressed by hTrx-1 transfection or pretreatment with rhTrx-1. Endogenous Trx-1 expression was induced by DEP in control cells. The downregulation of Akt phosphorylation by DEP resulted in apoptosis, which was prevented by Trx-1. Moreover, an Akt inhibitor canceled this protective effect of Trx-1. Collectively, the results suggest that Trx-1 exerts antioxidant effects in vivo and in vitro and that this plays a role in protection against DEP-induced lung damage by regulating Akt-mediated antiapoptotic signaling.     

Modification of potassium channel kinetics by histidine-specific reagents

We have examined the actions of histidine-specific reagents on potassium channels in squid giant axons. External application of 20-500 microM diethylpyrocarbonate (DEP) slowed the opening of potassium channels with little or no effect on closing rates. Sodium channels were not affected by these low external concentrations of DEP. Internal application of up to 2 mM DEP had no effect on potassium channel kinetics. Steady-state potassium channel currents were reduced in an apparently voltage-dependent manner by external treatment with this reagent. The shape of the instantaneous current-voltage relation was not altered. The voltage-dependent probability of channel opening was shifted toward more positive membrane potentials, thus accounting for the apparent voltage-dependent reduction of steady-state current. Histidine-specific photo-oxidation catalyzed by rose bengal produced alterations in potassium channel properties similar to those observed with DEP. The rate of action of DEP was consistent with a single kinetic class of histidine residues. In contrast to the effects on ionic currents, potassium channel gating currents were not modified by treatment with DEP. These results suggest the existence of a histidyl group (or groups) on the external surface of potassium channels important for a weakly voltage-dependent conformational transition. These effects can be reproduced by a simple kinetic model of potassium channels.     

Cellular basis of the role of diesel exhaust particles in inducing Th2-dominant response

There is growing evidence that diesel exhaust particles (DEP) can induce allergic diseases with increased IgE production and preferential activation of Th2 cells. To clarify the cellular basis of the role of DEP in the induction of Th2-dominant responses, we examined the effects of DEP on the cytokine production by T cells stimulated with anti-CD3/CD28 Ab and on that by monocyte-derived dendritic cells (MoDCs) stimulated with CD40L and/or IFN-gamma. We examined IFN-gamma, IL-4, IL-5, IL-8, and IL-10 produced by T cells and TNF-alpha, IL-1beta, IL-10, and IL-12 produced by MoDCs using real-time PCR analysis or by ELISA. To highlight the effects of DEP, we compared the effects of DEP with those of dexamethasone (DEX) and cyclosporin A (CyA). DEP significantly suppressed IFN-gamma mRNA expression and protein production, while it did not affect IL-4 or IL-5 mRNA expression or protein production. The suppressive effect on IFN-gamma mRNA expression was more potent than that of DEX and comparable at 30 mug/ml with 10(-7) M CyA. The suppressive effect on IFN-gamma production was also more potent than that of either DEX or CyA. DEP suppressed IL-12p40 and IL-12p35 mRNA expression and IL-12p40 and IL-12p70 production by MoDCs, while it augmented IL-1beta mRNA expression. Finally, by using a thiol antioxidant, N-acetyl cysteine, we found that the suppression of IFN-gamma production by DEP-treated T cells was mediated by oxidative stress. These data revealed a unique characteristic of DEP, namely that they induce a Th2 cytokine milieu in both T cells and dendritic cells.     

Histidine residues at the active site of maize δ-aminolevulinic acid dehydratase

Modification of maize δ-aminolevulinic acid dehydratase (ALAD) by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation showed saturation kinetics with a half inactivation time at saturating DEP equal to 0.3 min and K_DEP  0.3 mM. Substrate δ-aminolevulinic acid (ALA) and competitive inhibitor levulinic acid protected against inactivation, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.8 M hydroxylamine. Most of the activity lost by DEP treatment could be restored after treatment with 0.8 M hydroxylamine. The results suggest that DEP modifies 7.4 residues/mole of the enzyme. These histidine residues are essential for catalysis by ALAD.     

Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP

Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.     

Effect of Diethyl Pyrocarbonate Modification of Benzodiazepine Receptors on [3H]Ro 15-4513 Binding

The effects of treatment of brain membranes with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, on the binding of 3H-labeled Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]- [1,4]benzodiazepine-3-carboxylate) and [3H]diazepam were compared. DEP pretreatment produced a dose-dependent decrease in [3H]diazepam binding, whereas low DEP concentrations enhanced the binding of [3H]Ro 15-4513. These effects were reversed by incubation with hydroxylamine after the treatment. The enhancement of [3H]Ro 15-4513 binding was due to an increase in the affinity of the binding sites (KD), without any effect on binding capacity (Bmax). The enhancement was perceived in cerebral cortical, cerebellar, and hippocampal membranes. DEP treatment decreased the displacement of [3H]Ro 15-4513 binding by diazepam and FG 7142 (N-methyl-beta-carboline-3-carboxamide) but not by Ro 15-4513 and Ro 19-4603 (tert-butyl-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5- a]thieno[2,3-f][1,4]diazepine-3-carboxylate). Although the stimulating effect of gamma-aminobutyric acid (GABA) on [3H]-diazepam binding was not affected by DEP treatment, such treatment reduced the inhibitory effect of GABA on [3H]Ro 15-4513 binding. The enhancement of [3H]Ro 15-4513 binding was observed in membranes pretreated with DEP in the presence of flunitrazepam, whereas such pretreatment reduced significantly the inhibitory effect of DEP on [3H]-diazepam binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inflammatory and genotoxic effects of diesel particles in vitro and in vivo

Recent studies have identified an indirect genotoxicity pathway involving inflammation as one of the mechanisms underlying the carcinogenic effects of air pollution/diesel exhaust particles (DEP). We investigated the short-term effects of DEP on markers of inflammation and genotoxicity in vitro and in vivo. DEP induced an increase in the mRNA level of pro-inflammatory cytokines and a higher level of DNA strand breaks in the human lung epithelial cell line A549 in vitro. For the in vivo study, mice were exposed by inhalation to 20 or 80 mg/m3 DEP either as a single 90-min exposure or as four repeated 90-min exposures (5 or 20 mg/m3) and the effects in broncho-alveolar lavage (BAL) cells and/or lung tissue were characterized. Inhalation of DEP induced a dose-dependent inflammatory response with infiltration of macrophages and neutrophils and elevated gene expression of IL-6 in the lungs of mice. The inflammatory response was accompanied by DNA strand breaks in BAL cells and oxidative DNA damage and increased levels of bulky DNA adducts in lung tissue, the latter indicative of direct genotoxicity. The effect of a large single dose of DEP was more pronounced and sustained on IL-6 expression and oxidative DNA damage in the lung tissue than the effect of the same dose administered over four days, whereas the reverse pattern was seen in BAL cells. Our results suggest that the effects of DEP depend on the rate of delivery of the particle dose. The mutation frequency (MF), after DEP exposure, was determined using the transgenic Muta Mouse and a similar exposure regimen. No increase was observed in MF in lung tissue 28-days after exposure. In conclusion, short-term exposure to DEP resulted in DNA strand breaks in BAL cells, oxidative DNA damage and DNA adducts in lungs; and suggested that DNA damage in part is a consequence of inflammatory processes. The response was not associated with increased MF, indicating that the host defence mechanisms were sufficient to counteract the adverse effects of inflammation. Thus, there may be thresholds for the inflammation-associated genotoxic effects of DEP inhalation.     

Diesel exhaust particles suppress in vivo IFN-gamma production by inhibiting cytokine effects on NK and NKT cells

Diesel exhaust particles (DEP) have strong, selective Th2 adjuvant activity when inhaled with conventional Ags. We used a novel technique for measuring in vivo cytokine production to investigate possible mechanisms by which DEP might promote a Th2 response. Injection of DEP i.p. stimulated IL-6 secretion, but failed to increase IL-4, IL-10, or TNF-alpha secretion, and decreased basal levels of IFN-gamma. When injected with or before LPS, DEP had little effect on the LPS-induced TNF-alpha responses, but partially inhibited the LPS-induced IL-10 response and strongly inhibited the LPS-induced IFN-gamma response. DEP also inhibited the IFN-gamma responses to IL-12, IL-12 plus IL-18, IL-2, and poly(I.C). DEP treatment had little effect on the percentages of NK and NKT cells in the spleen, but inhibited LPS-induced IFN-gamma production by splenic NK and NKT cells. In contrast, DEP failed to inhibit the IFN-gamma response by anti-CD3 mAb-activated NKT cells. Taken together, these observations suggest that DEP inhibit Toll-like receptor ligand-induced IFN-gamma responses by interfering with cytokine signaling pathways that stimulate NK and NKT cells to produce IFN-gamma. Our observations also suggest that DEP may promote a Th2 response by stimulating production of inflammatory cytokines while simultaneously inhibiting production of IFN-gamma, and raise the possibility that the same mechanisms contribute to the association between DEP exposure and asthma.     

Diethyl phthalate, a chemotactic factor secreted by Helicobacter pylori.

The structure of a small-molecule, non-peptide chemotactic factor has been determined from activity purified to apparent homogeneity from Helicobacter pylori supernatants. H. pylori was grown in brucella broth media until one liter of solution had 0.9 absorbance units. The culture was centrifuged, and the bacteria re-suspended in physiological saline and incubated at 37 degrees C for 4 h. A monocyte migration bioassay revealed the presence of a single active chemotactic factor in the supernatant from this incubation. The chemotactic factor was concentrated by solid phase chromatography and purified by reverse phase high pressure liquid chromatography. The factor was shown to be indistinguishable from diethyl phthalate (DEP) on the basis of multiple criteria including nuclear magnetic resonance spectroscopy, electron impact mass spectroscopy, UV visible absorption spectrometry, GC and high pressure liquid chromatography retention times, and chemotactic activity toward monocytes. Control experiments with incubated culture media without detectable bacteria did not yield detectable DEP, suggesting it is bacterially derived. It is not known if the bacteria produce diethyl phthalate de novo or if it is a metabolic product of a precursor molecule present in culture media. DEP produced by H. pylori in addition to DEP present in man-made products may contribute to the high levels of DEP metabolites observed in human urine. DEP represents a new class of chemotactic factor.     

Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats

Pollution by particulates has consistently been associated with increased cardiorespiratory morbidity and mortality. It has been suggested that ultrafine particles, of which diesel exhaust particles (DEP) are significant contributors, are able to translocate from the airways into the bloodstream in vivo. In the present study, we assessed the effect of systemic administration of DEP on cardiovascular and respiratory parameters. DEP were administered into the tail vein of rats, and heart rate, blood pressure, blood platelet activation, and lung inflammation were studied 24 h later. Doses of 0.02, 0.1, or 0.5 mg DEP/kg (8, 42, or 212 microg DEP/rat) induced a significant decrease of heart rate and blood pressure compared with saline-treated rats. Although the number of platelets was not affected, all the doses of DEP caused a shortening of the bleeding time. Similarly, in addition to triggering lung edema, the bronchoalveolar lavage analysis revealed the presence of neutrophil influx in DEP-treated rats in a dose-dependent manner. We conclude that the presence of DEP in the systemic circulation leads not only to cardiovascular and haemostatic changes but it also triggers pulmonary inflammation.     

Theoretical and experimental study of the membrane pore effect on the amplitude-frequency dependence of polarizability of a cell

The amplitude-frequency dependence of the polarizability of erythrocytes, yeast cells, and latex particles in the range of 1–10⁶ Hz was studied by the method of dielectrophoresis (DEP). Positive DEP of erythrocytes and yeast cells in a frequency range of 60–100 Hz was revealed. The positive DEP of cells in the given range is theoretically explained by appearance of a great number of transverse pores through the membrane and wall of the cell.     

Separation of viable and nonviable animal cell using dielectrophoretic filter

Selective separation of cells using dielectrophoresis (DEP) has recently been studied and methods have been proposed. However, these methods are not applicable to large‐scale separation because they cannot be performed efficiently. In DEP separation, the DEP force is effective only when it is applied close to the electrodes. Utilizing a DEP filter is a solution for large‐scale separation. In this article, the separation efficiency for viable and nonviable cells in a DEP filter was examined. The effects of an applied AC electric field frequency and the gradient of the squared electric field intensity on a DEP velocity for the viable and nonviable animal cells (3‐2H3 cell) were discussed. The frequency response of the DEP velocity differed between the viable and the nonviable cells. We deducted an empirical equation that can be used as guiding principle for the DEP separation. The results indicate that the viable and the nonviable cells were separated using the DEP filter, and the best operating conditions such as the applied voltage and the flow rate were discussed. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The role of MAC1 in diesel exhaust particle‐induced microglial activation and loss of dopaminergic neuron function

Increasing reports support that air pollution causes neuroinflammation and is linked to central nervous system (CNS) disease/damage. Diesel exhaust particles (DEP) are a major component of urban air pollution, which has been linked to microglial activation and Parkinson's disease‐like pathology. To begin to address how DEP may exert CNS effects, microglia and neuron‐glia cultures were treated with either nanometer‐sized DEP (< 0.22 μM; 50 μg/mL), ultrafine carbon black (ufCB, 50 μg/mL), or DEP extracts (eDEP; from 50 μg/mL DEP), and the effect of microglial activation and dopaminergic (DA) neuron function was assessed. All three treatments showed enhanced ameboid microglia morphology, increased H₂O₂ production, and decreased DA uptake. Mechanistic inquiry revealed that the scavenger receptor inhibitor fucoidan blocked DEP internalization in microglia, but failed to alter DEP‐induced H₂O₂ production in microglia. However, pre‐treatment with the MAC1/CD11b inhibitor antibody blocked microglial H₂O₂ production in response to DEP. MAC1^?/? mesencephalic neuron‐glia cultures were protected from DEP‐induced loss of DA neuron function, as measured by DA uptake. These findings support that DEP may activate microglia through multiple mechanisms, where scavenger receptors regulate internalization of DEP and the MAC1 receptor is mandatory for both DEP‐induced microglial H₂O₂ production and loss of DA neuron function.     

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance.