Characterization of alveolar macrophage eicosanoid production in a non-human primate model of mineral dust exposure

The relative activation of eicosanoid production which results from the exposure of the alveolar macrophage (AM) to mineral dusts is thought to be a key factor in the pathophysiology of occupational lung disease. We compared in vitro basal and silica-stimulated production of prostaglandin E₂ (PGE₂) and thromboxane A₂ (TXA₂) by AM from normal humans and non-human primates (Macaca nemistrina). In addition, we instilled mineral dusts directly into one lung of the non-human primate and evaluated AM eicosanoid production at two week intervals following dust instillation. Unstimulated AM from humans produce more PGE₂ and TXA₂ than do AM from M. nemistrina. However, in vitro exposure of AM from both species to silica dust produced a qualitatively similar increase in TXA₂ production accompanied by no change in PGE₂ production. Sequential analysis of AM eicosanoid production following a single bolus exposure to bituminous or anthracite coal dusts, titanium dioxide (TiO₂) dust or crystalline silica showed marked variability among individual non-human primates in qualitative and quantitative aspects of dust-induced eicosanoid production. However, the rank order of potency of the different dusts (silica > anthracite > bituminous) correlated with epidemiological evidence relating the type of dust mined to the incidence of pneumoconiosis. These studies suggest that the non-human primate may serve as a model for the study of both the role of eicosanoids in the etiology of dust-induced occupational lung disease and the biochemical basis for individual variability in the response of lung cells to mineral dust exposure.     

Effect of allotype on activation of neutrophils by FcgammaRIIIB cross-linking

Human neutrophils constitutively synthesize two receptors for the constant region of IgG, FcgammaRII, and FcgammaRIIIB. Fluo-3-loaded neutrophils were treated with biotinylated Fab fragments of anti-FcgammaR antibodies and cross-linked with streptavidin, and intracellular calcium ([Ca2+](i)) was monitored by flow cytometry. Polymerization of filamentous actin was quantitated by NBD-phallacidin using flow cytometry. Cross-linking of FcgammaRII by monoclonal antibody (mAb) IV.3 induces an increase in [Ca2+](i), superoxide generation, and the polymerization of actin. [Ca2+](i) responses from cross-linking of FcgammaRIIIB by mAb 3G8 varied from minimal to no release. To determine whether discrepancies in 3G8-induced [Ca2+](i) release were due to allotype variation, we selected five donors who were homozygous for the NA1 allotype of FcgammaRIIIB and five who were either heterozygous or homozygous for the NA2 allotype and compared their [Ca2+](i) response and actin polymerization induced by FcgammaRIIIB cross-linking. Cross-linking of FcgammaRIIIB by 3G8 produced minimal [Ca2+](i) release and polymerization of actin irrespective of donor allotype.     

Apoptosis of lymphocytes induced by chromium(VI/V) is through ROS-mediated activation of Src-family kinases and caspase-3

Mechanistic insights into Cr(VI)-induced carcinogenicity and possible implication of Cr(V) species formed by the redox reactions of chromium-bearing species have attracted interest. We have previously demonstrated that when human peripheral blood lymphocytes are exposed to the Cr(V) complexes, viz., sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[Cr(V)O(ehba)(2)] and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr(V)O(hmba)(2)], apoptosis and formation of reactive oxygen species (ROS) are observed. The molecular mechanisms involving cellular signaling pathways leading to apoptosis are addressed in the present study. Treatment of lymphocytes with Na[Cr(V)O(ehba)(2)] and K(2)Cr(2)O(7) leads to the activation of the Src-family protein tyrosine kinases namely, p56(lck), p59(fyn), and p56/53(lyn), which then activates caspase-3, both of which are under the partial influence of ROS. Inhibition of the Src-family tyrosine kinases activity by PP2 and of caspase-3 by Z-DEVD-FMK reverses apoptosis, thereby suggesting their importance. Antioxidants only partially reverse the apoptosis induced by Cr(VI/V), suggesting that pathways other than those induced by ROS cannot be ruled out. Although the complex, Na[Cr(V)O(ehba)(2)] is known to be relatively stable in aqueous solutions, previous studies have shown that the Cr(V) complex, Na[Cr(V)O(ehba)(2)] disproportionates to Cr(VI) and Cr(III) forms at pH 7.4 through complex mechanistic processes. Dynamics studies employing EPR data show that the Cr(V) state in Na[Cr(V)O(ehba)(2)] is relatively more stable in RPMI-1640 medium containing plasma. Formation of ROS during the reaction of redox partners with Na[Cr(V)O(ehba)(2)] is an early event and compares favorably in kinetic terms with the reported rate processes for disproportionation. This investigation presents evidence for the direct implication of Cr(V) in Cr(VI)-induced apoptosis of lymphocytes.     

Calcium signaling is involved in cadmium-induced neuronal apoptosis via induction of reactive oxygen species and activation of MAPK/mTOR network

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+](i)) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+](i) elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+](i) elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+](i) elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+](i), which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+](i) homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.     

Inhibition of hypoxia-induced calcium responses in pulmonary arterial smooth muscle by acetazolamide is independent of carbonic anhydrase inhibition

Hypoxic pulmonary vasoconstriction (HPV) occurs with ascent to high altitude and can contribute to development of high altitude pulmonary edema (HAPE). Vascular smooth muscle contains carbonic anhydrase (CA), and acetazolamide (AZ), a CA inhibitor, blunts HPV and might be useful in the prevention of HAPE. The mechanism by which AZ impairs HPV is uncertain. Originally developed as a diuretic, AZ also has direct effects on systemic vascular smooth muscle, including modulation of pH and membrane potential; however, the effect of AZ on pulmonary arterial smooth muscle cells (PASMCs) is unknown. Since HPV requires Ca2+ influx into PASMCs and can be modulated by pH, we hypothesized that AZ alters hypoxia-induced changes in PASMC intracellular pH (pH(i)) or Ca2+ concentration ([Ca2+](i)). Using fluorescent microscopy, we tested the effect of AZ as well as two other potent CA inhibitors, benzolamide and ethoxzolamide, which exhibit low and high membrane permeability, respectively, on hypoxia-induced responses in PASMCs. Hypoxia caused a significant increase in [Ca2+](i) but no change in pH(i). All three CA inhibitors slightly decreased basal pH(i), but only AZ caused a concentration-dependent decrease in the [Ca2+](i) response to hypoxia. AZ had no effect on the KCl-induced increase in [Ca2+](i) or membrane potential. N-methyl-AZ, a synthesized compound lacking the unsubstituted sulfonamide group required for CA inhibition, had no effect on pH(i) but inhibited hypoxia-induced Ca2+ responses. These results suggest that AZ attenuates HPV by selectively inhibiting hypoxia-induced Ca2+ responses via a mechanism independent of CA inhibition, changes in pH(i), or membrane potential.     

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Reaction of cysteine and glutathione (GSH) at the freshly fractured quartz surface: a possible role in silica-related diseases?

The reactivity of quartz dusts towards glutathione (GSH) and cysteine (Cys) has been investigated. Cys and GSH react, without being adsorbed (UV-Vis spectroscopy), with commercial quartz dusts in an exposed surface-dependent way, but not with amorphous silica. GSH and Cys have been contacted with freshly ground quartz (agate jar QZg-a and steel jar QZg-s) and quartz heated in air at 500 degrees C (QZs-500) and with a dust generated from a purified quartz (99.9999%) to detect the nature of the reacting surface sites. With both GSH and Cys, the highest reactivity was found on the particles ground in a steel jar, while pure quartz was fully inactive. Detection of the radical GS* (spin trapping) suggests a radical mechanism of oxidation to disulphide onto surface-bound iron traces, more abundant on QZg-s and absent on the pure quartz. Oxidation of thiol groups occurs at surface sites different from those involved in the homolytic rupture of a C-H bond. Both reactions are more pronounced on freshly ground samples, but the C-H rupture takes place at silicon-based surface radicals and Fe2+ centers, while oxidation of GSH and Cys requires Fe3+ centers. As all commercial quartz dusts contain surface iron as an impurity, depletion of extracellular or intracellular GSH may contribute to the oxidative damage caused by particle-derived and cell-derived reactive oxygen species.     

Adsorption of lipoproteins onto mineral dust surfaces: a possible factor in the pathogenesis of particle-induced pulmonary fibrosis?

We compare the adsorption behavior of high density lipoproteins (HDL) and low density lipoproteins (LDL) on "fibrogenic" and "nonfibrogenic" mineral dusts. The adsorption tests with bovine lipoprotein concentrate and human serum produced the following results: 1) All seven examined fibrogenic dusts (SiO2 DQ12, SiO2 F600, silica, graphite, TiC, kaolin, talc) adsorbed significantly more high density lipoproteins (HDL), than the five examined nonfibrogenic (inert) dusts (TiO2, SnO2, Al2O3, Fe2O3, Fe3O4). This different behavior was particularly conspicuous in the presence of competing adsorbates (serum proteins). 2) In contrast, the adsorption of LDL did not correlate with the fibrogenicity of the mineral dusts. 3) The known silicosis-protective substance polyvinylpyridine-N-oxide inhibits the HDL adsorption of alpha-quartz. These results indicate that the adsorption of HDL could have a causal relationship with the triggering of a fibrotic reaction. The adsorption on the surface of fibrogenic dust particles provides an exceptional opportunity for the intake of HDL by macrophages. During the phagocytosis of the inhaled dust particles, the HDL adsorbed on the surface of the particles could be taken up by macrophages regardless of the receptor. There the HDL particles and/or compounds associated with them, such as lecithin-cholesterol-acyltransferase, could stimulate the macrophages to release fibrogenic mediators by some yet unknown mechanism.     

Crystalline silica Min-U-Sil 5 induces oxidative stress in human bronchial epithelial cells BEAS-2B by reducing the efficiency of antiglycation and antioxidant enzymatic defenses

Reactive oxygen species (ROS) play an important role as mediators of pulmonary damage in mineral dust-induced diseases. Studies carried out to date have largely focused on silica-induced production of ROS by lung phagocytes. In this study we investigated the hypothesis that crystalline silica Min-U-Sil 5 can induce elevations in intracellular ROS in human bronchial epithelial cells BEAS-2B, via an indirect mechanism that involves ROS-inducing intracellular factors, through a reduction of antiglycation (glyoxalase enzymes) and antioxidant (paraoxonase 1 and glutathione-S-transferases) enzymatic defenses. The results show that crystalline silica Min-U-Sil 5 causes a significant reduction in the efficiency of antiglycation and antioxidant enzymatic defenses, paralleled by an early and extensive ROS generation, thus preventing the cells from an efficient scavenging action, and eliciting oxidative damage. These results confirm the importance of ROS in development of crystalline silica-induced oxidative stress and emphasize the pivotal role of antiglycation/antioxidant and detoxifying systems in determining the level of protection from free radicals-induced injury for cells exposed to crystalline silica Min-U-Sil 5.     

The enzymatic removal of a surfactant coating from quartz and kaolin by P388D1 cells

The macrophage-like cell line, P388D1, was exposed to dipalmitoyl lecithin (DPL)-coated respirable quartz and kaolin, and the disappearance of the DPL was monitored for up to 9 days. The coating was removed rapidly at first (about 50% in the first 3 days) and then more slowly over the remaining 6 days, until about 30% remained on day 9. The rate of DPL digestion was independent of the type of dust and the amount of coated dust within the cell, indicating the existence of an extracellular phospholipase activity. This extracellular phospholipase activity was partially characterized. It was sensitive to temperatures above 56°C, the presence of EDTA, the action of the proteases trypsin and proteinase K, and pH, being active at pH 7 but not at pH 5. This is consistent with reports in the leterature of the existence of an extralysosomal phospholipase which is active at pH 7 and dependent on the presence of divalent metal ions. There was a dust-dependent difference in the extracellular rate of DPL digestion from quartz and kaolin. The coating was removed more slowly from the kaolin than it was from quartz. The removal of the DPL coating seen in the presence of cells was presumably due to both an intracellular and an extracellular phospholipase.Abbreviations ATCC American Type Culture Collection - BET Bruneur-Emmet-Teller - CP chlorphentermine - CQ chloroquine - DPL dipalmitoyl phosphatidyl lecithin - EDS energy-dispersive X-ray spectrometry - I imipramine - OsO₄ osmium tetroxide - PBS phosphate-buffered saline - FBS fetal bovine serum     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

Highly selective and rapid enrichment of phosphorylated peptides using gallium oxide-coated magnetic microspheres for MALDI-TOF-MS and nano-LC-ESI-MS/MS/MS analysis

In this work, we present, to our knowledge, the first demonstration of the utility of iron oxide magnetic microspheres coated with gallium oxide for the highly selective enrichment of phosphopeptide prior to mass spectrometric analysis. These microspheres that we prepared not only have a shell of gallium oxide, giving them a high-trapping capacity for the phosphopeptides, but also their magnetic property enables easy isolation by positioning an external magnetic field. Tryptic digest products of phosphoproteins including beta-casein, ovalbumin, casein, as well as five protein mixtures were used as the samples to exemplify the feasibility of this approach. In very short time (only 0.5 min), phosphopeptides sufficient for characterization by MALDI-TOF-MS were selectively enriched by the Ga(2)O(3)-coated Fe(3)O(4) microspheres. The performance of the Ga(2)O(3)-coated Fe(3)O(4) microspheres were further compared with Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC resin, and TiO2 beads for enrichment of peptides originating from tryptic digestion of beta-casein and BSA with a molar ratio of 1:50, and the results proved a stronger selective ability of Ga(2)O(3)-coated Fe(3)O(4) microspheres over the other materials. Finally, the Ga(2)O(3)-coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. All results show that Ga(2)O(3)-coated Fe(3)O(4) microsphere is an effective material for selective isolation and concentration of phosphopeptides.     

Oxidation of anthracycline anticancer agents by the peroxidase mimic microperoxidase 11 and hydrogen peroxide

The interaction of two clinically important anticancer agents doxorubicin (DXR) and daunorubicin (DNR) and the DNR analog 5-iminodaunorubicin (5IDNR) with the model mammalian peroxidase microperoxidase 11 (MP11) and H(2)O(2) has been investigated using spectrophotometric and EPR techniques. We demonstrate that DNR, DXR, and 5IDNR undergo irreversible oxidation by MP11/H(2)O(2), forming colorless products in both phosphate buffer pH 7.0 and in phosphate buffer pH 7.0/MeOH mixture (1:1 vol/vol), suggesting an extensive modification of the compounds' chromophores. The initial rate of the anthracyclines' oxidation is independent of anthracycline concentrations, but is linearly dependent on [H(2)O(2)](i) at constant [MP11](i) (and vice versa), indicating that the reaction is zero order in [anthracycline], first order with respect to [H(2)O(2)] and [MP11], and second order overall. Based on data obtained using DNR, DXR, 5IDNR, and p-hydroquinone k(2app), the apparent second order rate constant for the formation of a reactive intermediate from MP11 and H(2)O(2) (an analog of peroxidase compound I) has been determined to be in the range of (2.51-5.11) x 10(3) M(-1) s(-1) in both solvent systems. EPR studies show that oxidation of DNR, DXR, or 5IDNR with MP11/H(2)O(2) generates free radicals, suggesting that the reaction may be a one-electron process. This study also shows that 5IDNR, but not DNR or DXR, efficiently protects MP11 heme against degradation by H(2)O(2). Our overall conclusion is that MP11 is an effective catalyst of oxidation of anthracyclines by H(2)O(2). Given that, at sites of inflammation or cancer, the anthracyclines can colocalize with peroxidases, protein degradation products, and with H(2)O(2), peroxidation could be one possible fate of these anticancer agents in vivo.     

Mineral and chemical characteristics,textural parameters,and the mobility of the selected elements of flotation waste,originating from the Polish copper-mining industry

This article discusses the mineralogy and geochemical characteristics of the fresh copper-flotation waste samples. The mobility of As, Cd, Cr, Cu, Ni, Pb, Tl, Zn was investigated by leaching tests. The main mineral phases identified concerned dolomite, quartz, clay minerals, feldspars, and copper-bearing minerals. Chemically, CaO and silica were dominating, along with a significant concentration of precious (Cu), refractory (Cr, Ti, V, Zr), and toxic (As, Cd, Pb) metals. Elements were bound mainly to the residual fraction and sulphides in the following order: Pb > Cu ≈ Tl > As ≈ Zn > Ni ≈ Cr > Cd. The metal mobility patterns expressed as a percentage of total concentrations, were as follows: Cd (42%) > Cr (26%)> Ni (24%) > Zn (23%) > As (22%) > Tl (20%) > Cu (18%) > Pb (2%). Those constituents were released earlier in lower pH values, although Cu, Cr, and Pb were also released in higher alkaline pH values. However, Zn release was not dependent on pH. When L/S values decreased, elements like As, Cr, Cu, Pb, and Tl were released. That process caused decrease of Cd, Ni, and Zn release.     

Pulmonary vascular response to normoxia and K(Ca) channel activity is developmentally regulated

To address developmental regulation of pulmonary vascular O(2) sensing, we tested the hypotheses that 1) fetal but not adult pulmonary artery smooth muscle cells (PASMCs) can directly sense an acute increase in O(2), 2) Ca2+-sensitive K(+) (K(Ca)) channel activity decreases with maturation, and 3) PASMC K(Ca) channel expression decreases with maturation. We used fluorescence microscopy to confirm that fetal but not adult PASMCs are able to sense an acute increase in O(2) tension. Acute normoxia induced a 22 +/- 2% decrease in cytosolic Ca2+ concentration ([Ca2+](i)) in fetal PASMCs and no change in ([Ca2+](i)) in adult PASMCs (P < 0.01). The effects of K(+) channel antagonists were studied on fetal and adult PASMC ([Ca2+](i)). Iberiotoxin (10(-9) M) caused PASMC ([Ca2+](i)) to increase by 694 +/- 22% in the fetus and caused no change in adult PASMCs. K(Ca) channel expression and mRNA levels in distal pulmonary arteries from fetal and adult sheep were examined. Both K(Ca) channel protein and mRNA expression in the distal pulmonary vasculature decreased with maturation. We conclude that maturation-dependent changes in PASMC O(2) sensing render the fetal PASMCs uniquely sensitive to an acute increase in O(2) tension at a biologically critical time point.     

Chemical prevention of colony foundation by Cryptotermes brevis (Isoptera: Kalotermitidae) in attic modules

Disodium octaborate tetrahydrate (DOT) dust, DOT aqueous solution, imidacloprid dust, and amorphous silica gel dust with synergized 1% pyrethrins were applied on wood surfaces to simulated attic modules. Modules (30 by 30 cm) with and without fiberglass insulation were exposed to dispersal flights of Cryptotermes brevis (Walker) in May and June of 1998 and 1999. Six months after flights, modules were disassembled and inspected for nuptial chamber location and contents. During both years, air and water control treatments contained 22.2+/-9.94 (mean +/- SD) nuptial chambers, 7.5+/-5.7 live imagos, and 2.0+/-1.4 chambers with brood. This survivorship indicated that the attic modules performed well as a colonizing platform for C. brevis. C. brevis dealates preferred constructing nuptial chambers in the crevices at the bases or tops of the modules instead of internal crevices. Modules treated in 1998 and 1999 with DOT or silica dusts contained no live termites, whereas zero of five modules treated with imidacloprid dust in 1998 and two of 20 modules treated with imidacloprid dust in 1999 contained single live incipient colonies. In 1998, 15% DOT solution, applied as a postconstruction treatment, yielded significantly fewer chambers and live termites than controls, but was not as effective as dusts in preventing successful colonization. In 1999, the DOT solution, applied as a construction-phase treatment, was equally as effective in preventing colonization as the dust treatments during that year. Results indicate that dust formulations of DOT, silica gel, and imidacloprid can be used to prevent drywood termite colonization in existing building voids and attics. Where the entire wood framing is exposed to treatment, such as during building construction, aqueous DOT solution can be equally effective as dusts in preventing colonization by C. brevis.     

Rapid degradation of PrxI and PrxII induced by silica in Rat2 cells

Peroxidases of the peroxiredoxin (Prx) family catalyze the reduction of H(2)O(2) and lipid peroxides. The effects of H(2)O(2), 12-O-tetradecanoylphorbol 13-acetate (TPA), and silica on the abundance of two cytosolic isoforms of Prx (PrxI and PrxII) were examined in Rat2 cells. TPA induces the production of reactive oxygen species (ROS) in various mammalian cell types, and silica induces the production of ROS in Rat2 cells. Whereas H(2)O(2) and TPA did not affect the concentration of PrxI or Prx II, silica triggered a rapid degradation of both Prx enzymes. Silica also induced degradation of the NF-kappaB inhibitor IkappaB-alpha. N-Acetylcysteine and diphenyleneiodonium, both of which inhibit the accumulation of intracellular ROS, each blocked silica-induced degradation of IkappaB-alpha but had no effect on that of the Prx enzymes, suggesting that ROS do not contribute to Prx proteolysis. The silica-induced degradation of Prx enzymes was also insensitive to the proteasome inhibitors MG132 and lactacystin, whereas IkappaB-alpha proteolysis was completely blocked by these inhibitors. Experiments with the Ca(2+) ionophore A23187 indicated that a Ca(2+)-dependent protease such as calpain might contribute substantially to silica-induced degradation of PrxII, but only moderately to that of PrxI. These results indicate that silica increases cellular oxidative stress not only by inducing ROS production, but also by triggering the degradation of Prx enzymes that are responsible for elimination of cellular ROS. Such aggravated oxidative stress might be important in the initial pathogenesis of silica-associated pulmonary diseases.     

Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction

Cell lines expressing varying levels of ganglioside GM3 at the cell surface show different degrees of adhesion and spreading on solid phase coated with such glycosphingolipids (GSLs) as Gg3 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1Cer), LacCer (Gal beta 1----4Glc beta 1----1Cer), or Gb4 (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer) (where Cer is ceramide), which may have structures complementary to GM3, but not on solid phase coated with various other GSLs. The degree of cell adhesion and spreading on Gg3 was correlated with the degree of cell-surface GM3 expression, as defined by reactivity with anti-GM3 monoclonal antibody (mAb) DH2. Only cells with high GM3 expression adhered on solid phase coated with LacCer or Gb4. Adhesion of GM3-expressing cells on Gg3-, LacCer-, and Gb4-coated solid phase is based on interaction of GM3 with Gg3 and, to a lesser extent, with LacCer and Gb4, as demonstrated by: (i) the interaction of the GM3 liposome with solid phase coated with Gg3, LacCer, and Gb4, respectively; (ii) the abolition of cell adhesion on each GSL-coated solid phase by treatment of cells with mAb DH2 or sialidase; and (iii) the inhibition of cell adhesion by treatment of GSL-coated solid phase with mAb specific to each GSL. Sialosyllactosyl-lysyllysine conjugate was bound to Gg3 adsorbed on a C18 silica gel column in the presence of bivalent cation, suggesting that the carbohydrate moiety of GM3 is involved in GM3-Gg3 interaction. Not only the adhesion and spreading of GM3-expressing cells, but also their cell motility was greatly enhanced on Gg3-coated solid phase, as determined by Transwell assay and phagokinetic track assay on a gold sol-coated surface. Spreading and motility of GM3-expressing cells on Gg3-coated solid phase were both inhibited by treatment of cells with mAb DH2 or sialidase. These results provide evidence that not only cell adhesion, but also spreading and motility in these cell lines are controlled by complementary GSL-GSL interaction.     

Mechanical stress stimulates phospholipase C activity and intracellular calcium ion levels in neonatal rat cardiomyocytes

To investigate how mechanical stress is sensed by cardiomyocytes and translated to cardiac hypertrophy, cardiomyocytes were subjected to stretch while measuring phospholipase C (PLC) and phospholipase D (PLD) activities and levels of intracellular calcium ions ([Ca2+]i) and pH.In stretched cardiomyocytes, PLC activity increased 2-fold after 30 min, whereas PLD activity hardly increased at all. Mechanical stress induced by prodding or by cell stretch increased [Ca2+](i)by a factor 5.2 and 4, respectively. Gadolinium chloride (stretch-activated channel blocker) attenuated the prodding-induced and stretch-induced [Ca2+](i)rise by about 50%. Blockade of ryanodine receptors by a combination of Ruthenium Red and procaine reduced the [Ca2+](i)rise only partially. Diltiazem (L-type Ca2+ channel antagonist) blocked the prodding-induced [Ca2+](i)rise completely, and reduced the stretch-induced [Ca2+](i)rise by about 50%. The stretch-induced [Ca2+](i)rise was unaffected by U73122, an inhibitor of PLC activity. Stretch did not cause cellular alkalinization.In conclusion, in cardiomyocytes, PLC and [Ca2+](i)levels are involved in the stretch-induced signal transduction, whereas PLD plays apparently no role. The stretch-induced rise in [Ca2+](i)in cardiomyocytes is most probably caused by [Ca2+](i)influx through L-type Ca2+ channels and stretch-activated channels, leading to Ca2+-induced Ca2+ -release from the SR via the ryanodine receptor.     

Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 microM) and NBQX (10 microM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase--oligomycin, an uncoupler of oxidative phosphorylation--CCCP, a chelator of intracellular Ca2+--BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport--ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+](i). Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.