Detection of stratospheric ozone intrusions by windprofiler radars.

Stratospheric ozone attenuates harmful ultraviolet radiation and protects the Earth's biosphere. Ozone is also of fundamental importance for the chemistry of the lowermost part of the atmosphere, the troposphere. At ground level, ozone is an important by-product of anthropogenic pollution, damaging forests and crops, and negatively affecting human health. Ozone is critical to the chemical and thermal balance of the troposphere because, via the formation of hydroxyl radicals, it controls the capacity of tropospheric air to oxidize and remove other pollutants. Moreover, ozone is an important greenhouse gas, particularly in the upper troposphere. Although photochemistry in the lower troposphere is the major source of tropospheric ozone, the stratosphere-troposphere transport of ozone is important to the overall climatology, budget and long-term trends of tropospheric ozone. Stratospheric intrusion events, however, are still poorly understood. Here we introduce the use of modern windprofiler radars to assist in such transport investigations. By hourly monitoring the radar-derived tropopause height in combination with a series of frequent ozonesonde balloon launches, we find numerous intrusions of ozone from the stratosphere into the troposphere in southeastern Canada. On some occasions, ozone is dispersed at altitudes of two to four kilometres, but on other occasions it reaches the ground, where it can dominate the ozone density variability. We observe rapid changes in radar tropopause height immediately preceding these intrusion events. Such changes therefore serve as a valuable diagnostic for the occurrence of ozone intrusion events. Our studies emphasize the impact that stratospheric ozone can have on tropospheric ozone, and show that windprofiler data can be used to infer the possibility of ozone intrusions, as well as better represent tropopause motions in association with stratosphere-troposphere transport.

Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology.

Proteome analysis is most commonly accomplished by a combination of two-dimensional gel electrophoresis (2DE) to separate and visualize proteins and mass spectrometry (MS) for protein identification. Although this technique is powerful, mature, and sensitive, questions remain concerning its ability to characterize all of the elements of a proteome. In the current study, more than 1,500 features were visualized by silver staining a narrow pH range (4.9-5. 7) 2D gel in which 0.5 mg of total soluble yeast protein was separated. Fifty spots migrating to a region of 4 cm(2) were subjected to MS protein identification. Despite the high sample load and extended electrophoretic separation, proteins from genes with codon bias values of <0.1 (lower abundance proteins) were not found, even though fully one-half of all yeast genes fall into that range. Proteins from genes with codon bias values of <0.1 were found, however, if protein amounts exceeding the capacity of 2DE were fractionated and analyzed. We conclude that the large range of protein expression levels limits the ability of the 2DE-MS approach to analyze proteins of medium to low abundance, and thus the potential of this technique for proteome analysis is likewise limited.

Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.

Several methods have been developed for the comprehensive analysis of gene expression in complex biological systems. Generally these procedures assess either a portion of the cellular transcriptome or a portion of the cellular proteome. Each approach has distinct conceptual and methodological advantages and disadvantages. We have investigated the application of both methods to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cells. This pathway is of critical importance for the development and progression of prostate cancer. Of clinical importance, modulation of androgens remains the mainstay of treatment for patients with advanced disease. To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate-derived expressed sequence tags (ESTs). A total of 55000 prostate ESTs were assembled into a set of 15953 clusters putatively representing 15953 distinct transcripts. These clusters were used to construct cDNA microarrays suitable for examining the androgen-response pathway at the level of transcription. The expression of 20 genes was found to be induced by androgens. This cohort included known androgen-regulated genes such as prostate-specific antigen (PSA) and several novel complementary DNAs (cDNAs). Protein expression profiles of androgen-stimulated prostate cancer cells were generated by two-dimensional electrophoresis (2-DE). Mass spectrometric analysis of androgen-regulated proteins in these cells identified the metastasis-suppressor gene NDKA/nm23, a finding that may explain a marked reduction in metastatic potential when these cells express a functional androgen receptor pathway.

Analysis of integrin (CD11b/CD18) movement during neutrophil adhesion and migration on endothelial cells.

Little is known of the distribution of cell surface molecules during the adhesion and migration of leucocytes on endothelial cells. We have used confocal microscopy and a Fab fragment of a non-inhibitory monoclonal antibody recognizing the integrin CD11b/CD18 (Mac-1) to study the movement of this adhesion molecule over time. We found that during the initial stage of neutrophil contact with TNF-alpha activated human umbilical vein endothelial cells (HUVEC), there is a rapid accumulation of Mac-1 at the contact area between the two cell types. As the neutrophil spreads, Mac-1 redistributes away from this initial contact area. During neutrophil migration on HUVEC, Mac-1 was redistributed to the leading edge of the migrating cell, suggesting that the existing cell surface pool of adhesion molecules is dynamic and can be recruited to the leading front as the cell changes direction. As neutrophils migrate on HUVEC, Mac-1-dense macroaggregates are rapidly formed and broken down at the contact plane between the two cells. The confocal microscope, coupled with the use of non-inhibitory antibodies labelled with photostable fluorophores, is a useful tool for the study of the movement of cell surface molecules over time.

Correlation between protein and mRNA abundance in yeast.

We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast Saccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243-251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.

Binding of human peripheral blood polymorphonuclear leukocytes to E-selectin (CD62E) does not promote their activation.

E-selectin (CD62E) is a cytokine-inducible endothelial cell adhesion molecule that tethers polymorphonuclear leukocytes (PMNs) and supports PMN rolling under conditions of flow. We examined whether interaction of PMNs with E-selectin also leads to activation of CD11b/CD18 (Mac-1, alphaMbeta2), an event that can promote firm adhesion. PMNs were added to monolayers of IL-1beta-activated HUVECs and Chinese hamster ovary (CHO) cells transfected with E-selectin cDNA. PMN activation was assessed by 1) increased CD11b/CD18 surface expression, 2) appearance of activation epitope on CD11b/CD18 (CD11b*) detected by mAb CBRM1/5, and 3) decreased L-selectin (CD62L) expression, as determined by flow cytometry. Both adherent and nonadherent supernatant PMNs became activated on IL-1beta-pretreated HUVECs. This activation was not affected by CD62E-blocking mAb P6E2. The activation state of PMNs adhered to CHO cells transfected with E-selectin cDNA was not increased over background and was similar to that of PMNs exposed to parent CHO cells. The findings were confirmed using confocal microscopy, which allowed staining of the cells for CD11b* in situ. In concert, the results suggest that PMN binding to E-selectin does not elicit inter-receptor signaling that could result in strengthening of PMN adhesion to endothelium.

Blockade of CD49d (alpha4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma.

Immunized mice after inhalation of specific antigen have the following characteristic features of human asthma: airway eosinophilia, mucus and Th2 cytokine release, and hyperresponsiveness to methacholine. A model of late-phase allergic pulmonary inflammation in ovalbumin-sensitized mice was used to address the role of the alpha4 integrin (CD49d) in mediating the airway inflammation and hyperresponsiveness. Local, intrapulmonary blockade of CD49d by intranasal administration of CD49d mAb inhibited all signs of lung inflammation, IL-4 and IL-5 release, and hyperresponsiveness to methacholine. In contrast, CD49d blockade on circulating leukocytes by intraperitoneal CD49d mAb treatment only prevented the airway eosinophilia. In this asthma model, a CD49d-positive intrapulmonary leukocyte distinct from the eosinophil is the key effector cell of allergen-induced pulmonary inflammation and hyperresponsiveness.

Western blot assay for prostate-specific membrane antigen in serum of prostate cancer patients.

There is a need for the development of new diagnostic tools for the early detection of prostate cancer. A candidate molecule for a new screening test is a prostate-specific membrane antigen (PSM) recognized by the monoclonal antibody 7E11.C5. We carried out studies aimed at identifying PSM in the serum of normal and benign prostatic hyperplasia (BPH) donors and patients with adenocarcinoma of the prostate, in order to judge whether the development of a serum assay using this marker was feasible. By Western blotting, we found significant levels of PSM in serum samples from prostatic cancer patients, in the seminal fluid of pooled normal donors, in BPH patients, and in normal male sera. Similar to prostate-specific antigen (PSA), PSM was present in seminal plasma in higher concentrations than in serum, and PSM levels in prostatic cancer patients were significantly higher than in normal controls. These data suggest that the development of an assay utilizing the PSM and new monoclonal antibodies directed against the antigen, could provide a feasible test for prostatic cancers.

Lipopolysaccharide enhances CD11b/CD18 function but inhibits neutrophil aggregation.

Human neutrophils are primed in the presence of complexes of lipopolysaccharide (LPS) with its serum binding protein (LBP) in a manner dependent on CD14. Cellular consequences of priming include increased responsiveness, the upregulation of surface proteins including the adhesive integrin CD11b/CD18 (Mac-1), the increased binding of certain ligands to CD11b/CD18, and the concurrent shedding of the L-selectin homing receptor. Because expression of both CD11b/CD18 and L-selectin is obligatory for formyl peptide-stimulated neutrophil aggregation in vitro (Simon et al, Blood 82:1097, 1993), we have examined the consequences of bacterial endotoxin on the expression of neutrophil adhesive molecules. We observed that the exposure of neutrophils to LPS/LBP, while enhancing the surface numbers and adhesive function of CD11b/CD18 for latex particles, did not induce aggregation. In contrast, as the LPS/LBP concentration increased (ED50 = 30 ng/mL LPS/LBP), the ability of neutrophils to aggregate decreased in parallel with the shedding of L-selectin. Moreover, when L-selectin adhesive activity was blocked by treatment with Fab fragments of Dreg-200, aggregation was inhibited to an extent roughly proportional to the available L-selection. Blocking of LPS/LBP with CD14-specific monoclonal antibodies suppressed L-selectin shedding and preserved formyl peptide-stimulated aggregation. Taken together, the data suggest that inhibition of neutrophil aggregation by LPS/LBP is related to the expression of L-selectin via CD14 rather than LPS inhibition of CD11b/CD18 function during cellular stimulation.

A role for lectin interactions during human neutrophil aggregation.

We have recently reported that neutrophil aggregation is dependent on both L-selectin and the beta 2-integrin Mac-1, raising the possibility that carbohydrate interactions play a role in aggregation. We used mono- and polysaccharides known to inhibit L-selectin-dependent adhesion of lymphocytes to high endothelial venules to test whether these carbohydrates could inhibit neutrophil aggregation. Similar types and concentrations of carbohydrates found by others to inhibit lymphocyte adhesion were effective in blocking neutrophil aggregation. Thus, nanomolar concentrations of the polysaccharides dextran sulfate (m.w. 500,000) and fucoidan inhibited aggregation, whereas dermatan sulfate, alpha-carrageenan, and dextran sulfate (m.w. 5,000) showed no inhibition. All of the phosphorylated monosaccharides tested inhibited aggregation with ED50 values between 8 and 17 mM, the most potent being mannose-6-phosphate and fucose-1-phosphate. The nonphosphorylated monosaccharides glucose and fucose were noninhibitory. The inhibitory effects of fucoidan or dextran sulfate (m.w. 500,000) did not appear to be due to altered regulation of L-selectin after stimulation because fucoidan reduced the rate of L-selectin shedding, whereas dextran sulfate had no effect compared with control. Neither carbohydrate inhibited the binding of formyl peptide to its receptor. However, carbohydrates were able to compete with mAb binding to a number of known leukocyte adhesion proteins. We used endotoxin pretreatment to create L-selectin-deficient neutrophils to study the minimum adhesive requirements for aggregation using two-color fluorescence flow cytometry. Our results implicate a lectinlike contribution to neutrophil aggregation, and suggest that L-selectin is the molecule that mediates the carbohydrate-dependent adhesive event.

Regulation of human neutrophil aggregation: comparable latent times, activator sensitivities, and exponential decay in aggregability for FMLP, platelet-activating factor, and leukotriene B4.

We have recently described a flow cytometry technique, whose sensitivity allows direct measurements of latent times before the onset of aggregation, and of rates, maximal extents, and reversibility of aggregation (J Leuk Biol 50:434, 1991). We report here that activators which stimulate sustained cellular signaling associated with increases in intracellular calcium (ionomycin) or protein kinase C activation (phorbol myristate acetate, PMA) cause complete (> or = 98%) and irreversible neutrophil aggregation, with latent times for the onset of aggregation inversely proportional to the activator concentration. In contrast, the receptor-specific activators leukotriene B4 (LTB4), formyl peptide FMLP, and platelet-activating factor (PAF) gave only partial and reversible aggregatory responses, limited by the following similar properties: latent times of 4.5 seconds +/- 1.5 seconds, independent of activator concentration; similar concentrations for onset of aggregation (approximately 1 nmol/L) that increased over a similar broad range of activator concentration, with one-half maximal rates of aggregation at 10 nmol/L to 30 nmol/L, corresponding to reported dissociation constant values; comparable limited recruitment and spontaneous reversibility of aggregation; absence of interactivator synergism; and similar exponential decays in activated cell stickiness (refractoriness), with t1/2 = 15 to 30 seconds. Variable cross-desensitization was seen between LTB4 and FMLP depending on donor and activator concentrations. In vivo, these properties are expected to provide localization of the aggregatory response, minimizing the otherwise detrimental effects of circulating activated neutrophils.

Beta 2-integrin and L-selectin are obligatory receptors in neutrophil aggregation.

We have recently found that antibodies to L-selectin, the homing receptor on neutrophils, are as effective as those to beta 2-integrin at blocking formyl peptide-stimulated aggregation. Therefore, we investigated the requirements for expression of L-selectin and beta 2-integrin on adjacent cells during aggregation. Fluorescence flow cytometry allowed characterization of aggregates on the basis of size and color, as well as antibody binding to these two adhesive molecules. Formyl peptide-stimulated aggregate formation was measured for individual populations fluorescently labeled red (LDS-751) or green (CD44-FITC), and interpopulation red-green cell conjugates. Blocking either the beta 2-integrin or L-selectin adhesive epitope with monoclonal antibody on individual cell populations resulted in an approximately 50% reduction in two-color aggregation as compared with that in unblocked samples. Shedding the L-selectin on a cell population by preincubation with complexes of lipopolysaccharide and its plasma membrane binding protein also decreased aggregation to a control population by approximately 50%. We examined the aggregation of neutrophils from patients genetically deficient in beta 2-integrin and clinically leukocyte adhesion deficient (LAD). LAD adhesion to normal neutrophils was dependent on the expression of L-selectin on LAD cells and beta 2-integrin on normal cells. Thus, the minimum requirement for adhesion between two mixed populations of neutrophils was that one population expressed the beta 2-integrin and the other expressed the L-selectin adhesive epitope.

A model for the recruitment of neutrophils at sites of inflammation. Physiological relevance of in vivo neutrophil aggregation.

Adhesive glycoproteins on both neutrophils and vascular endothelial cells are known to mediate adhesive interaction between the two cell types. We propose that activation of endothelial cells will lead to the capture of unactivated neutrophils, localizing them at inflammatory sites. The interaction between activated endothelium and captured neutrophils will result in the stimulation of adherent neutrophils. Adherent activated neutrophils are then able to recruit incoming unactivated neutrophils by capturing them, further increasing the number of neutrophils at the inflammatory site. The formation of an adherent neutrophil aggregate will reduce plasma leakage from the vasculature as neutrophils migrate into tissue and will protect migrating neutrophils from shear stress of blood flow.

Dynamics of human neutrophil aggregation evaluated by flow cytometry.

In order to directly monitor neutrophil aggregation, we have developed a simple particle counting technique using flow cytometry. Flow times were used to determine aggregation from changes in the total number of particles per unit volume (%PA(T)), while fluorescent signals emitted from glutaraldehyde-fixed neutrophils were used to measure changes in the total number of singlet neutrophils (%PA(S)). Flow cytometrically-determined %PA values were found to be virtually identical to values determined from microscopy. We show that aggregation parameters can be evaluated and compared simply from measures of changes in total particle count without a need for distinguishing singlets from multiplets. A number of aggregation parameters are introduced and related to the initial cell concentration (N(o)) and stir speed (shear). Aggregation of neutrophils, following stimulation with 0.5 microM FMLP (N-formyl-methionyl-leucyl-phenylalanine), showed a latent time (tl) of 4 +/- 1.5 sec independent of N(o) or stir speed; had a forward rate of aggregation (vf) which was proportional to the initial cell concentration and the stir speed; plateaued for greater than or equal to 60 s; and showed partial to complete reversal by 7.5 min. Above a critical stir speed, the extent and duration of aggregation varied inversely with the stir speed. The stir and N(o) dependence of the aggregation parameters studied suggest the existence of subpopulations of neutrophils with distinct efficiencies of aggregation.

Comparative studies of microscopically determined aggregation, degranulation, and light transmission after chemotactic activation of adult and newborn neutrophils.

Changes in the light transmission of suspensions of activated neutrophils are widely used to measure the dynamics of neutrophil aggregation. Such studies have suggested, for example, that aggregation is irreversible for human newborn neutrophils but fully reversible for adult cells. We have evaluated aggregation directly by microscopic particle counting and compared it with changes in light transmission (delta T) and with release from three granule subsets for neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP). Maximal increases in %T in response to 0.5 micromol/L FMLP were approximately 25% larger for newborn than for adult neutrophils, and were only partially reversible by 8 minutes, while %T increases for adult neutrophils were fully reversible. However, measurements of neutrophil aggregation using light microscopy showed that both newborn and adult neutrophils fully deaggregated. A further independence of delta T from aggregation was found by pretreating adult neutrophils with cytochalasin B (5 micrograms/mL) in the presence of 0.5% gelatin, a pretreatment that blocked FMLP-induced neutrophil aggregation while allowing large increases in %T and degranulation. In response to FMLP, newborn neutrophils released more enzyme from each granule subset than did adult neutrophils. Our results suggest that cellular events associated with neutrophil activation (other than aggregation) are implicated in light transmission responses and that these differ for adults and newborns. These results also suggest that reports of neutrophil aggregation should be based on direct particle counting methods rather than on %T responses.

Synergistic inhibition of complement induced granulocyte margination by BW755C and calcium channel blockers.

Intravenous infusion of granulocyte (PMNL) chemotactic factors including C5ades Arg present in zymosan activated plasma (ZAP), induces granulocytopenia due to PMNL margination. Since some PMNL responses are dependent on Ca++ ions and lipoxygenation of arachidonic acid, we evaluated the effects of a lipoxygenase (and cyclooxygenase) inhibitor, BW755C and Ca++ channel blocking agents, verapamil and nifedipine, on chemotactic factor induced granulocytopenia and margination in rabbits. BW755C (20 mg/kg i.v.) treatment significantly attenuated ZAP induced granulocytopenia. Verapamil or nifedipine alone were without effect. However, combined treatment with BW755C and verapamil or nifedipine (250 micrograms/kg) completely prevented ZAP-induced granulocytopenia. Ibuprofen, a cyclooxygenase inhibitor, was without effect either by itself or in combination with the calcium channel blockers. In striking contrast to the effect on ZAP-induced granulocytopenia, BW755C plus verapamil or nifedipine had virtually no effect on f-met-leu-phe, platelet activating factor or leukotriene B4 induced granulocytopenia. PMNL aggregation in vitro in response to all of the above chemotactic factors was inhibited by BW775C to similar degrees (56-75%) and was not influenced by simultaneous treatment with verapamil. We conclude that: (a) inhibitors of the lipoxygenase pathway may synergize with Ca++ channel blocking agents in inhibiting PMNL responses to complement derived chemotactic factors in vivo; (b) that in vivo PMNL margination to other chemotactic factors may be less dependent on endogenous lipoxygenation and/or Ca++ fluxes; and (c) there is a poor correlation between pharmacological inhibition of PMNL aggregation in vitro and PMNL margination in vivo in this system.

A role for hemolysin in Escherichia coli-induced inflammation in granulocytopenic rabbits.

Inflammation induced in the skin of granulocytopenic rabbits by Escherichia coli was examined. Protein exudation and platelet deposition in lesions were measured with 125I-labeled albumin and 111In-labeled platelets. In granulocytopenic rabbits 10(4)-10(7) live serum-resistant E. coli induced protein exudation and platelet deposition beginning at 3 hr and then progressing over the next 24 hr to much higher levels than in normal rabbits. These responses were associated with interstitial edema and progressive venous thrombosis in the absence of leukocytes; no such reactions were observed in normal rabbits. No reactions were induced in granulocytopenic rabbits by killed E. coli. Of six E. coli strains tested, all three hemolytic strains induced lesions with four to five times more thrombosis (platelet deposition) than did nonhemolytic strains. Two hemolysin-negative mutants lost most of their thrombogenic activity. All three hemolytic strains had cell-associated hemolysin, but only one of these elaborated appreciable free, filterable hemolysin as well.

Formation of ursodeoxycholic acid from chenodeoxycholic acid by a 7 beta-hydroxysteroid dehydrogenase-elaborating Eubacterium aerofaciens strain cocultured with 7 alpha-hydroxysteroid dehydrogenase-elaborating organisms.

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2