Resonant tunneling through electronic trapping states in thin MgO magnetic junctions.

We report an inelastic electron tunneling spectroscopy study on MgO magnetic junctions with thin barriers (0.85-1.35 nm). Inelastic electron tunneling spectroscopy reveals resonant electronic trapping within the barrier for voltages V>0.15 V. These trapping features are associated with defects in the barrier crystalline structure, as confirmed by high-resolution transmission electron microscopy. Such defects are responsible for resonant tunneling due to energy levels that are formed in the barrier. A model was applied to determine the average location and energy level of the traps, indicating that they are mostly located in the middle of the MgO barrier, in accordance with the high-resolution transmission electron microscopy data and trap-assisted tunneling conductance theory. Evidence of the influence of trapping on the voltage dependence of tunnel magnetoresistance is shown.

Caffeic acid phenethyl ester prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration.

Parkinson's disease is associated with the loss of dopaminergic neurons in the substantia nigra and decreased striatal dopamine levels. We now report that caffeic acid phenethyl ester (CAPE), an active component of propolis, attenuated dopaminergic neurodegeneration and dopamine loss in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease. The neuroprotective effect of CAPE was associated with marked reductions in inducible nitric oxide synthase (iNOS) and caspase 1 expression. Additionally, CAPE inhibited MPP+-induced neurotoxicity in vitro and directly inhibited MPP+-induced release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Thus, CAPE may have beneficial effects in slowing or preventing the progression of Parkinson's disease and other neurodegenerative disorders.

Vitronectin protects alveolar macrophages from silica toxicity.

Silicosis is an interstitial lung disease caused by the inhalation of crystalline silicon dioxide. Current concepts suggest that a crucial step in the development of silicosis is silica-induced injury of alveolar macrophages (AM). The adhesive protein vitronectin is a natural constituent of the lung, in which its function is largely unexplored. This study investigated a possible role for vitronectin in protecting AM from silica exposure. In this study, the concentration of vitronectin was shown to be increased in the bronchoalveolar lavage fluid of silica-treated rats. Vitronectin affinity for silica was shown both in vitro and in vivo by immunostaining. Vitronectin reduced silica-induced injury to cultured AM as determined with the (51)Cr release assay. Vitronectin reduced silica-induced free radical production as determined with a cell-free thiobarbituric acid assay. Additionally, vitronectin reduced the silica-induced respiratory burst in AM as determined with chemiluminescence. This study suggests that vitronectin may protect AM during the initial exposure to silica.

Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages.

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a (51)Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 +/- 2.5, whereas the addition of rat SP-A (5 microg/ml) significantly reduced the cytotoxic index to 16.6 +/- 1.2 (P < 0. 001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody or D-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.

Nonimmune phagocytosis of liposomes by rat alveolar macrophages is enhanced by vitronectin and is vitronectin-receptor mediated.

Pulmonary alveolar macrophages (AMs) engulf diverse materials. The mechanisms allowing AMs to recognize, bind, and phagocytose these materials are poorly understood. To test the hypothesis that the adhesive glycoprotein vitronectin (Vn) acts as a nonimmune opsonin, we studied AM-Vn binding and AM phagocytosis of fluorescent liposomes under the following conditions: (1) pretreatment of AMs with Vn, followed by incubation of AMs with liposomes containing increased amounts of Vn; (2) inhibition of phagocytosis by gly-arg-gly-asp-ser (RGD) and gly-pen-gly-arg-gly-asp-ser-pro-cys-ala (GPen); and (3) antibody blockade of the alpha(v)beta3 vitronectin receptor (VnR). Pretreatment of AMs with 0.1, 1, and 2 microM Vn progressively enhanced AM-Vn binding from 23,622 +/- 3,328 cpm to 40,847 +/- 6,530 cpm, 57,149 +/- 2,789 cpm, and 124,852 +/- 42,930 cpm, respectively (P < 0.05). AM pretreatment also increased phagocytosis of Vn-enriched liposomes, but not empty liposomes (20.7 +/- 0.4 liposomes/cell versus 11.5 +/- 0.5 liposomes/cell, P < 0.05). Moreover, increased concentrations of Vn in liposomes progressively increased phagocytic activity (3.7 +/- 0.3, 6.5 +/- 0.2, 11.5 +/- 0.5, and 16.5 +/- 0.6 liposomes/cell with 0.01, 0.1, and 1 microM Vn, respectively, P < 0.05). RGD inhibited Vn-enhanced phagocytosis (8.1 +/- 0.4 liposomes/cell to 3.4 +/- 0.2, 2.4 +/- 0.4, and 2.2 +/- 0.2 liposomes/cell with 0.02, 0.2, and 2 mM RGD, respectively, P < 0.05), as did GPen (4.7 +/- 0.8 liposomes/cell versus control = 10.9 +/- 1.5 liposomes/cell, P < 0.05) and anti-VnR antibody (3.3 +/- 0.4 liposomes/cell versus control = 8.9 +/- 1.7 liposomes/cell, P < 0.05). We conclude that AMs employ Vn as a nonimmune opsonin to enhance the efficiency of phagocytosis.

Assessment of in vivo attachment/phagocytosis by alveolar macrophages.

Alveolar macrophages (AMs) are recognized as an important first line of cellular host defense within the lung. Although mechanisms underlying AM response to microorganisms or particulates are well characterized in vitro, experimental approaches to the study of AMs in vivo are limited. To circumvent these limitations, a new assay was developed using fluorescently labelled liposomes or Pneumocystis carinii (PC) organisms which were administered intratracheally into mechanically ventilated rats. After 30 min, the lungs were lavaged and the percentage of administered liposomes or PC bound to AMs was determined by quantifying fluorescence. Factors known to enhance attachment/phagocytosis by AMs in vitro were assayed to determine their effect in vivo. For example, vitronectin (VN)-coated liposomes increased attachment from 25.2 +/- 2.4% to 47.2 +/- 3.0% (p < 0.001), while addition of VN increased the binding of PC to AMs from 16.5 +/- 1.7% to 24.5 +/- 2.2% (p < 0.05). Confocal laser microscopy of cells obtained by lavage provided morphologic evidence of attachment/phagocytosis by AMs. This model will permit the quantitative assessment of the interaction of fluorescently labelled liposomes or microorganisms with AMs in the lower respiratory tract of living animals.

Interaction of vitronectin with Pneumocystis carinii: evidence for binding via the heparin binding domain.

Pneumocystis carinii pneumonia is a major opportunistic infection in patients with the acquired immune deficiency syndrome. P. carinii attachment to alveolar epithelial cells is considered necessary for growth and replication of the organism. Recent studies have focused on the role of adhesive proteins such as fibronectin and vitronectin in attachment mechanisms of P. carinii in the alveolar space. Whereas the role of fibronectin has been partially characterized, less is known about the mechanism of vitronectin interaction with P. carinii. To better understand the mechanism underlying this interaction, vitronectin-P. carinii binding was characterized with respect to monovalent and divalent cations and pH by using an iodine 125-labeled vitronectin binding assay to P. carinii. As an example, vitronectin-P. carinii binding was abolished in the presence of 1.0 mol/L NaCl and enhanced by Ca2+ and Mn2+. Further, periodate and heparin treatment of P. carinii significantly reduced vitronectin binding to the organism to 10% +/- 1.5% (p < 0.01) and 52% +/- 1.8% (p < 0.05) of control values, respectively. There was no competitive inhibition of vitronectin binding to P. carinii by using the peptide sequence arg-gly-asp-ser of the cell binding domain. The findings suggest that vitronectin, unlike fibronectin, binds to P. carinii by a predominantly electrostatic mechanism that likely involves the heparin binding domain of vitronectin.

Pneumocystis carinii attachment increases expression of fibronectin-binding integrins on cultured lung cells.

Pneumocystis carinii is an extracellular pathogen which requires attachment to alveolar epithelial cells for growth and replication. Previous studies have demonstrated that the extracellular matrix protein fibronectin (Fn) facilitates attachment of P. carinii to lung cells. This study addresses the role of cell surface Fn receptors (integrins) as mediators of P. carinii attachment and demonstrates the effect of P. carinii attachment on integrin expression on cultured lung cells. To determine the role of Fn-binding integrins in P. carinii attachment, attachment of 51Cr-labelled P. carinii organisms to the lung epithelial cell line A549 was quantified in the presence or absence of anti-integrin antibodies. Antibodies to the alpha v and alpha 5 integrin subunits significantly inhibited P. carinii attachment, while addition of antibody to the alpha subunit of a non-Fn-binding integrin, alpha 2, did not affect P. carinii attachment. To further investigate the role of Fn-binding integrins in P. carinii attachment, the effect of P. carinii attachment on expression of the alpha v and alpha 5 integrin subunits was determined. A549 cells incubated with either P. carinii organisms or with the P. carinii major surface glycoprotein gp120 demonstrated a 3- to 10-fold increase in expression of the alpha 5 integrin subunit; however, neither P. carinii nor gp120 affected the expression of alpha v integrin. Furthermore, the effects of P. carinii on A549 cell alpha 5 integrin expression were attenuated by the addition of an anti-gp120 antibody which blocks P. carinii attachment to A549 cells. Therefore, P. carinii attachment to lung epithelial cells appears to be mediated by alpha v- and alpha 5-containing integrins expressed on the epithelial cell surface, and P. carinii attachment results in increased expression of the alpha 5 integrin subunit.

Isolation of Pneumocystis carinii gp120 by fibronectin affinity: evidence for manganese dependence.

Pneumocystis carinii is a major opportunistic lung pathogen and a leading cause of death among patients with the human immunodeficiency virus. Adherence of P. carinii to type I alveolar epithelial cells is essential for growth and replication and has been shown to be mediated in part by fibronectin (Fn). To better understand the mechanisms underlying this attachment, P. carinii-Fn interaction was characterized with respect to divalent and monovalent ion concentration and pH using an 125I-Fn binding assay to P. carinii. The results suggest that P. carinii has a receptor for Fn that was partially dependent on Ca2+, enhanced by Mn2+, and diminished somewhat by Mg2+. Additional data demonstrated that P. carinii-Fn interaction was sensitive to ionic strength. The pH profile revealed that P. carinii-Fn interaction increased with decreasing pH. The results from the binding assay provided the rationale for a simple isolation of the Fn receptor from P. carinii using a Fn-affinity column involving nondenaturing conditions. The isolated receptor appeared highly purified by SDS-PAGE analysis, with apparent molecular weights of 114 to 118 kD and 66 kD. Western blot analysis indicated that this receptor was gp120, a major surface glycoprotein of P. carinii. Furthermore, the isolated receptor inhibited Fn binding to P. carinii. Finally, a monoclonal antibody raised against the affinity-purified gp120 blocked Fn binding to P. carinii.

Fast and sensitive laser-based enzymatic detection of the lactose operon in microorganisms.

This work investigates the ability of laser-based, time-resolved fluorometry to detect the lactose operon genetic marker in microorganisms and to study protein-DNA interactions. In the first study, rapid detection of the Escherichia coli lacZY operon inserted in two strains of Pseudomonas proposed as fungal biological control organisms was achieved. Optimization of incubation time, immobilization apparatus size, and reagent volumes, along with the laser-based instrumentation, yielded an assay capable of detecting 10(4) immobilized lac+ Pseudomonas fluorescens cells within a 30-min incubation time. In the second study, the synthesis of E. coli beta-galactosidase was monitored in "real-time" with observable enzymatic activity beginning 4 to 5 min after induction with isopropylthiogalactoside.