Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-gamma) or TNF-alpha could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membrane-impermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-gamma- and TNF-alpha-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M(r) 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthase-beta, alpha-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.     

A consensus tetrapeptide selected by phage display adopts the conformation of a dominant discontinuous epitope of a monoclonal anti-VWF antibody that inhibits the von Willebrand factor-collagen interaction

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.     

The amount of free corticosterone is increased during lipopolysaccharide-induced fever

The relation between lipopolysaccharide (LPS)-induced fever and bioavailability of corticosterone (B) was examined in male Wistar rats. Animals were injected with LPS (2.5 mg/kg i.p.) or saline and core temperature and heart rate were monitored continuously using a biotelemetry system. Blood samples were withdrawn from freely moving rats via jugular catheters for estimation of total and free plasma B. LPS induced a long-lasting increase (24-48 h) in core temperature and B secretion and a short-lasting increase (90 min) in heart rate. LPS-induced fever was accompanied by a significant increase in the free/total B ratio. In contrast, an acute injection of B, which resulted in circulating B levels similar to those found after LPS, did not affect the free/total B ratio. The important role of LPS-induced fever in the hormone secretion pattern and the equilibrium between free and total B was further demonstrated in an in vitro study showing that an increase in the temperature by 3 degrees C elevated the free B fraction and the free/total B ratio of plasma samples with concentrations of B in the physiological range (5-40 microg/dl). Taken together, these findings indicate that during LPS-induced fever there is an increase in the amount of biologically available B. Exposure of glucocorticoid-sensitive targets to elevated levels of free B could contribute to the restoration of homeostasis that is disturbed during inflammation.     

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.The cytotoxic cell granule-secretory pathway depends on perforin (PFN) to deliver granzyme (Gzm) proteases to the cytosol of target cells where they induce apoptosis and other biological effects, such as inflammation.¹ Ring-shaped transmembrane PFN pores hereafter called ‘cylindrical pores'', are presumed to act as the gateway for cytosolic entry, either at the plasma membrane or after endocytosis.^{2, 3, 4} In either case the highly cationic Gzms are thought to diffuse through these cylindrical pores formed by poly-PFN. Nevertheless, a mechanistic understanding of the phenomenon (how the cationic globular protein exchanges from its carrier proteoglycan, serglycin, to the pore and crosses the plasma and/or vesicular membranes) has been lacking due to limitations in imaging technology and in our detailed understanding of the molecular forms that PFN may adopt following interaction with a target cell plasma membrane.Here we show under conditions where cylindrical pore formation is minimal,⁵ that granzyme B (GzmB) translocation readily occurs. We previously demonstrated that a prelude to granzyme translocation is PFN-mediated, Ca-independent phosphatidylserine (PS) externalization (flip-flop) measured by annexin-V and lactadherin binding.⁶ This rapid PS flip-flop also occurs when mouse CD8 cells contact antigen-pulsed target cells. Inasmuch as the proteinaceous cylinders offer a formidable barrier to lipid flow, we have speculated that the observed movement of anionic phospholipids to the external leaflet is due to the formation of proteo-lipidic structures, which consists of oligomerized PFN monomers bearing an arc morphology and plasma membrane lipids.^{6, 7, 8}In the work reported here, the topology of PFN embedded into homogeneous planar bilayers and tumor cell plasma membranes was imaged by atomic force microscopy (AFM) and deep etch electron microscopy (DEEM), respectively. Further, the influence of an anti-human PFN mAb (pf-80) that rescues target cells from necrosis,⁹ was examined. The AFM data show that PFN forms arcs as well as rings in planar bilayers, while conductance measurements across equivalent membranes in parallel experiments measured functional pore sizes consistent with these varied structures. The pf-80 mAb increased the frequency of arc formation and reduced conductance values. Interestingly, PS flip-flop and granzyme delivery were both increased in target cells after PFN oligomerization was interrupted by the pf-80 mAb. A similar effect was seen in T24 bladder carcinoma cells imaged by DEEM. Treatment with PFN leads to deposition of rings (barrel stave pores) and arcs and the pf-80 mAb increased the ratio of arcs to rings on the surface of these cells. We suggest that the observed protein arcs function as toroidal pores in whole cells, explaining PS flip-flop, and act as focal points for granzyme translocation across lipid bilayer.     

Aerobic fitness and performance in elite female futsal players

Despite its growing popularity, few studies have investigated specific physiological demands for elite female futsal. The aim of this study was to determine aerobic fitness in elite female futsal players using laboratory and field testing. Fourteen female futsal players from the Venezuelan National team (age =21.2±4.0 years; body mass =58.6±5.6 kg; height =161±5.0 cm) performed a progressive maximal treadmill test under laboratory conditions. Players also performed a progressive intermittent futsal-specific field test for endurance, the Futsal Intermittent Endurance Test (FIET), until volitional fatigue. Outcome variables were exercise heart rate (HR), VO₂, post-exercise blood lactate concentrations ([La]b) and running speeds (km · h^-1). During the treadmill test, VO₂max, maximal aerobic speed (MAS), HR and peak [La]b were 45.3±5.6 ml · kg^-1 · min^-1, 12.5±1.77 km · h^-1, 197±8 beats · min^-1 and 11.3±1.4 mmol · l^-1, respectively. The FIET total distance, peak running velocity, peak HR and [La]b were 1125.0±121.0 m, 15.2±0.5 km · h^-1, 199±8 beats · min^-1 and 12.5±2.2 mmol · l^-1, respectively. The FIET distance and peak speed were strongly associated (r= 0.85-87, p < 0.0001) with VO₂max and MAS, respectively. Peak HR and [La]b were not significantly different between tests. Elite female futsal players possess moderate aerobic fitness. Furthermore, the FIET can be considered as a valid field test to determine aerobic fitness in elite level female futsal players.     

Species-dependent differences of the biochemical properties of diamine oxidase

Diamine oxidase (DAO) from tissues of mice, rats and humans showed different properties with respect to stability and kinetic parameters. DAO-activities in homogenates of rat or human tissues, but not of mouse tissues, rapidly decreased upon storage at -20 degrees C. The Km-value for putrescine was 90 microM in mouse kidney or intestine. In rats different Km-values were observed before (272 microM) and after freezing (102 microM). A similar effect was observed with DAO in human kidney (321 and 39 microM, respectively). Treatment of rats with heparin resulted in a depletion of intestinal DAO and the concomitant appearance of DAO in blood. The enzyme remaining in the intestine showed the lower Km-value.     

Substrate metabolism during different exercise intensities in endurance-trained women.

We have studied eight endurance-trained women at rest and during exercise at 25, 65, and 85% of maximal oxygen uptake. The rate of appearance (R(a)) of free fatty acids (FFA) was determined by infusion of [(2)H(2)]palmitate, and fat oxidation rates were determined by indirect calorimetry. Glucose kinetics were assessed with [6,6-(2)H(2)]glucose. Glucose R(a) increased in relation to exercise intensity. In contrast, whereas FFA R(a) was significantly increased to the same extent in low- and moderate-intensity exercise, during high-intensity exercise, FFA R(a) was reduced compared with the other exercise values. Carbohydrate oxidation increased progressively with exercise intensity, whereas the highest rate of fat oxidation was during exercise at 65% of maximal oxygen uptake. After correction for differences in lean body mass, there were no differences between these results and previously reported data in endurance-trained men studied under the same conditions, except for slight differences in glucose metabolism during low-intensity exercise (Romijn JA, Coyle EF, Sidossis LS, Gastaldelli A, Horowitz JF, Endert E, and Wolfe RR. Am J Physiol Endocrinol Metab 265: E380-E391, 1993). We conclude that the patterns of changes in substrate kinetics during moderate- and high-intensity exercise are similar in trained men and women.     

T lymphocyte insensitivity to corticosteroids in chronic obstructive pulmonary disease

Background

There are increased numbers of activated lymphocytes in the lungs of chronic obstructive pulmonary disease (COPD) patients. The clinical benefits of corticosteroids in COPD patients are limited. Our hypothesis is that lymphocytes play a role in this corticosteroid insensitivity.

Objectives

To investigate the effects of the corticosteroid dexamethasone on lung lymphocyte cytokine production from patients with COPD compared to controls.

Methods

Cultured airway lymphocytes obtained by bronchoscopy from healthy non-smokers (HNS), smokers (S) and COPD patients were stimulated with phytohaemagglutinin (PHA) & phorbol myristate acetate (PMA), +/- dexamethasone. Supernatants were assayed for interleukin (IL)-2 and interferon (IFN)γ. Immunofluoresence was used to analyse changes in CD8 glucocorticoid receptor (GRα and GRβ) expression.

Results

The inhibition of PHA/PMA stimulated IFNγ production by dexamethasone was reduced in COPD patients compared to HNS (p < 0.05 at concentrations from 0.1-1 μM). There was also a significant reduction (p < 0.05) in the mean inhibitory effect at 1 μM in COPD patients (54.1%) compared to smokers (72.1%), and in smokers compared to HNS (85.5%). There was a numerically reduced effect of dexamethasone on IL-2 production that did not reach statistical significance. There was no difference in GRα and GRβ expression in follicular CD8 cells between COPD patients (50.9% and 30.4% respectively) and smokers (52.9% and 29.7% respectively).

Conclusions

IFNγ production from COPD airway lymphocytes is corticosteroid insensitive. This phenomenon may be important in the poor clinical response often observed with corticosteroids.     

Defunct brain stem cardiovascular regulation underlies cardiovascular collapse associated with methamphetamine intoxication

Background  

Intoxication from the psychostimulant methamphetamine (METH) because of cardiovascular collapse is a common cause of death within the abuse population. For obvious reasons, the heart has been taken as the primary target for this METH-induced toxicity. The demonstration that failure of brain stem cardiovascular regulation, rather than the heart, holds the key to cardiovascular collapse induced by the pesticide mevinphos implicates another potential underlying mechanism. The present study evaluated the hypothesis that METH effects acute cardiovascular depression by dampening the functional integrity of baroreflex via an action on brain stem nuclei that are associated with this homeostatic mechanism.     

Aspirin reduces hypertriglyceridemia by lowering VLDL-triglyceride production in mice fed a high-fat diet

Systemic inflammation is strongly involved in the pathophysiology of the metabolic syndrome, a cluster of metabolic risk factors that includes hypertriglyceridemia. Aspirin treatment lowers inflammation via inhibition of NF-κB activity but also reduces hypertriglyceridemia in humans. The aim of this study was to investigate the mechanism by which aspirin improves hypertriglyceridemia. Human apolipoprotein CI (apoCI)-expressing mice (APOC1 mice), an animal model with elevated plasma triglyceride (TG) levels, as well as normolipidemic wild-type (WT) mice were fed a high-fat diet (HFD) and treated with aspirin. Aspirin treatment reduced hepatic NF-κB activity in HFD-fed APOC1 and WT mice, and in addition, aspirin decreased plasma TG levels (-32%, P < 0.05) in hypertriglyceridemic APOC1 mice. This TG-lowering effect could not be explained by enhanced VLDL-TG clearance, but aspirin selectively reduced hepatic production of VLDL-TG in both APOC1 (-28%, P < 0.05) and WT mice (-33%, P < 0.05) without affecting VLDL-apoB production. Aspirin did not alter hepatic expression of genes involved in FA oxidation, lipogenesis, and VLDL production but decreased the incorporation of plasma-derived FA by the liver into VLDL-TG (-24%, P < 0.05), which was independent of hepatic expression of genes involved in FA uptake and transport. We conclude that aspirin improves hypertriglyceridemia by decreasing VLDL-TG production without affecting VLDL particle production. Therefore, the inhibition of inflammatory pathways by aspirin could be an interesting target for the treatment of hypertriglyceridemia.