An inelastic neutron scattering investigation of holmium orthoferrite.

The inelastic neutron scattering spectra recorded in this study and elsewhere provide a useful set of crystal-field (CF) energy levels for the groundJ= 6 term of Ho3+in HoFeO3. The resolution of the low-energy, temperature-dependent pseudo-quadrupole ground state splitting and magnon peaks is consistent with the self-ordering of the Ho3+sublattice atTHo∼ 8-10 K and supports earlier electron spin resonance investigations of the Ho3+magnon behaviour. Systematic analysis of the grouped singlet CF levels of Ho3: HoFeO3, in conjunction with the CF Kramers doublet levels of the neighbouring Er3+: ErFeO3, has yielded possible sets of CF parameters for the two systems.

Sequence analysis of cDNA coding for a major house dust mite allergen, Der p 1. Homology with cysteine proteases.

A cDNA clone coding for Der p 1, a major allergen from the house dust mite Dermatophagoides pteronyssinus, has been sequenced. It codes for a 222 residue mature protein with a derived molecular weight of 25,371 and contains 1 potential N-glycosylation site. In addition, the cDNA appears to code for a 13 residue proregion, and an incomplete signal peptide. The deduced sequence shows a high degree of homology with animal and plant cysteine proteases, particularly in the region of the contact residues making up the active site. Southern analysis of genomic DNA indicates that the allergen is coded by a noncontiguous gene. These data will now facilitate epitope mapping studies.

Reformation of functional liver polyribosomes from ribosome monomers in the absence of RNA synthesis.

The administration to rats of the ethyl analog of methionine, ethionine, results in the rapid decrease in the hepatic concentration of adenosine triphosphate followed by an extensive disaggregation of polysomes to ribosome monomers and a concomitant inhibition of protein synthesis. These effects are readily reversed by the injection of methionine or precursors of adenine nucleotides such as adenine. The reformation of liver polyribosomes in such animals following the administration of adenine plus methionine was found to occur under conditions in which new RNA synthesis was markedly inhibited. Free messenger RNA without attached ribosomes must be capable of remaining functionally active in the liver cytoplasm for many hours.

The crystal field interaction at the rare earth site in ErNiAl(4).

Inelastic neutron scattering spectra are reported for orthorhombic ErNiAl(4) at temperatures ranging from 2.1 to 280 K. The neutron transitions are interpreted in terms of three reliably identified excited crystal field (CF) levels and four tentative excited levels for the J = 7/2 ground term of Er(3+) at the single Er site. A shift in transition peak energy between 2.1 and 8.6 K is attributed to Zeeman splitting induced by magnetic order of the Er sub-lattice below T(N) = 5.8 K. With the aid of a suite of possible (155)Gd-Mössbauer spectroscopy derivations of the rank n = 2 CF parameters and a simple point charge model calculation of within-rank ratios for the higher rank (n = 4,6) parameters, estimates are made for all nine CF parameters required for the orthorhombic C(2v) (mm) Er-site symmetry.

The crystal chemistry of ferric oxyhydroxyapatite.

Ferric hydroxyapatites (Fe-HAp) and oxyapatites (Fe-OAp) of nominal composition [Ca(10-x)Fe(x)(3+)][(PO(4))(6)][(OH)(2-x)O(x)] (0 < or = x < or = 0.5) were synthesized from a coprecipitated precursor calcined under flowing nitrogen. The solid solubility of iron was temperature-dependent, varying from x = 0.5 after firing at 600 degrees C to x approximately 0.2 at 1000 degrees C, beyond which Fe-OAp was progressively replaced by tricalcium phosphate (Fe-TCP). Crystal size (13-116 nm) was controlled by iron content and calcination temperature. Ferric iron replaces calcium by two altervalent mechanisms in which carbonate and oxygen are incorporated as counterions. At low iron loadings, carbonate predominantly displaces hydroxyl in the apatite channels (Ca(2+) + OH(-) --> Fe(3+) + CO(3)(2-)), while at higher loadings, "interstitial" oxygen is tenanted in the framework (2Ca(2+) + (vac) --> 2Fe(3+) + O(2+)). Although Fe(3+) is smaller than Ca(2+), the unit cell dilates as iron enters apatite, providing evidence of oxygen injection that converts PO(4) tetrahedra to PO(5) trigonal bipyramids, leading to the crystal chemical formula [Ca(10-x)Fe(x)][(PO(4))(6-x/2)(PO(5))(x/2)][(OH)(2-y)O(2y)] (x < or = 0.5). A discontinuity in unit cell expansion at x approximately 0.2 combined with a substantial reduction of the carbonate FTIR fingerprint shows that oxygen infusion, rather than tunnel hydroxyl displacement, is dominant beyond this loading. This behavior is in contrast to ferrous-fluorapatite where Ca(2+) --> Fe(2+) aliovalent replacement does not require oxygen penetration and the cell volume contracts with iron loading. All of the materials were paramagnetic, but at low iron concentrations, a transition arising from crystallographic modification or a change in spin ordering is observed at 90 K. The excipient behavior of Fe-OAp was superior to that of HAp and may be linked to the crystalline component or mediated by a ubiquitous nondiffracting amorphous phase. Fe-HAp and Fe-OAp are not intrinsically suitable magnetic agents for drug delivery but may be useful in reactive cements that promote osteoblast proliferation.

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.

Magnetic structure and spin reorientation of quaternary Dy2Fe2Si2C.

We have investigated the low temperature magnetic properties of Dy2Fe2Si2C by using magnetisation, specific heat, x-ray diffraction, neutron powder diffraction and 57Fe Mössbauer spectroscopy measurements over the temperature range 1.5 K-300 K. Dy2Fe2Si2C exhibits two magnetic transitions at low temperatures: an antiferromagnetic transition at [Formula: see text] K and a spin-reorientation transition at [Formula: see text] K. The magnetic structure above T t can be described with a propagation vector [Formula: see text] with the ordering of the Dy magnetic moments along the monoclinic b-axis whereas on cooling below T t the Dy moment tips away from the b-axis towards the ac-plane. We find that the spin-reorientation in Dy2Fe2Si2C is mainly driven by the competition between the second-order crystal field term B 20 and the higher-order terms, in particular B 40 and B 64.

Glutathione peroxidase activity and mRNA expression in eosinophils and neutrophils of asthmatic and non-asthmatic subjects.

Asthma has been reported to be associated with a reduction in the activity of glutathione peroxidase (GSH-Px), an important antioxidant enzyme. However, the expression of GSH-Px enzyme activity has not previously been investigated in human eosinophils, which are important inflammatory cells involved in asthma. Reverse transcriptase-polymerase chain reaction and Southern blotting demonstrated that eosinophils express GSH-Px mRNA and the relative expression of GSH-Px was greater in eosinophils than in neutrophils for both asthmatic and non-asthmatic subjects. The presence of GSH-Px protein in eosinophil and neutrophil lysates was confirmed by size exclusion chromatography and by Western blotting. GSH-Px enzyme activity as measured by a spectrophotometric assay was greater in eosinophil (48.4+/-1.6 micromol NADPH oxidized x min(-1) x g(-1) protein) than in neutrophil lysates (18.1+/-0.4, n = 24, P < 0.0001). GSH-Px activities of eosinophils and neutrophils from asthmatic subjects did not differ from those of non-asthmatic subjects. Eosinophil GSH-Px activity was correlated with peripheral blood eosinophil count only in asthmatic subjects (rs = 0.59, n = 12, P = 0.04). Increased GSH-Px expression in eosinophils compared with neutrophils of asthmatic patients may provide antioxidant protection against the greater amounts of reactive oxygen species generated by these cells and may enhance the survival of eosinophils at sites of inflammation in asthma.

Immunogenicity and antigenicity of synthetic peptides derived from the mite allergen Der p I.

As an immunogen must contain both B- and T-cell epitopes, small peptides are usually reported as non-immunogenic unless coupled to a protein carrier. In this study, the immunogenicity of the Der p I synthetic uncoupled peptides (p52-71, p89-104, p117-133 and p176-187) previously reported as B-cell epitopes, was evaluated. Different schedules of immunization were used. Results indicated that by using the Vaitukaikis' method three injections of the same peptide without protein carrier was sufficient to induce an specific anti-peptide IgG antibody response (evaluated by ELISA). Indeed, the 16-20 amino-acid long peptides p52-71, p117-133 and p89-104 were revealed highly immunogenic in rabbits. Furthermore anti-peptide p52-71 and p117-133 antibodies were shown by Western-blotting or by neutralization assay to recognize the Der p I molecule either in denaturated or native form as well as Der f I (major allergen of Dermatophagoides farinae). Finally, taking into account the location of Der p I-derived peptides in the three-dimensional model of Der p I, the antigenicity and immunogenicity of peptides were discussed.

Production of multilamellar, small unilamellar and reverse-phase liposomes containing house dust mite allergens. Potential adjuvants in the immunotherapy of allergic disease.

The capacity of multilamellar (MLV), small unilamellar (SUV) and reverse-phase vesicle (REV) liposomes to incorporate house dust mite allergens has been studied. All three liposome preparations entrapped mite proteins with efficiencies of 36% (SUV), 29% (MLV) and 14% (REV). MLV incorporated the complete range of proteins contained in mite extracts with apparent molecular weights ranging from 14,000 to greater than 67,000 as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis. However, several proteins with apparent molecular weights (MW) of 16,000, 36,000 and 43,000 were excluded from the REV and SUV. Immunoblotting analysis using a serum pool prepared from mite allergic individuals showed that whereas the whole spectrum of allergens was incorporated into the MLV, a MW 43,000 allergen was excluded from the REV and SUV. The exclusion of these mite components is probably a function of the relatively prolonged exposure of the original extract to organic solvent in the preparation of the REV and SUV liposomes.

Empirical and theoretical relationships between the sedimentation coefficient and molecular weight of various proteins, with particular reference to the immunoglobulins.

The empirical and theoretical relationships between the sedimentation coefficient and molecular weight of proteins of various conformational shapes have been investigated. In each case it was found that the relationship obeys the general formula log S020,W= a log molecular weight +b. The empirical values obtained for the slope and intercept for globular proteins (non-immunoglobulins) and immunoglobulins have been used to calculate the molecular weight from the sedimentation coefficients of other proteins belonging to these two groups. For most proteins and particularly the immunoglobulins the values obtained were found to be similar to those obtained using the classical experimental techniques. The application of this approach to the determination of molecular weights, of proteins and more particularly protein containing complexes is discussed.

Antibodies to Dermatophagoides pteronyssinus allergen Dpt 12 contaminating rabbit antisera to human and mouse anti-immunoglobulins.

Antibodies to the allergen Dpt 12 from Dermatophagoides pteronyssinus have been demonstrated in commercially obtained rabbit anti-human IgE, IgG and IgA antisera and in a commercially obtained rabbit anti-mouse IgG1 antiserum using either double or triple antibody radioimmunoassays. Where measurable, the titre of anti-Dpt 12 antibodies varied both between different anti-isotypes and between batches of the same anti-isotype as judged by the dilution of antiserum required to bind 50% of the radiolabelled Dpt 12. The titres ranged from 1/23-1/360. In contrast, a specific rabbit anti-Dpt antiserum gave a titre of 1/4500.

ELISA plaque assay for the detection of antibody secreting cells: observations on the nature of the solid phase and on variations in plaque diameter.

This report examines the basis for variations in the size and number of ELISA plaques detected in vitro. The nature and concentration of antigen used to coat the solid phase is shown to be critical, high concentrations of polymerized antigen being optimal for low molecular weight proteins, while coating conditions for high molecular weight multideterminant antigens are similar to conventional liquid ELISA. Variations in plaque diameter are shown to reflect maturational changes in the immune response, and probably indicate differences in the affinity of antibody being secreted within a population of plasma cells.

Modulation of airway smooth muscle tone by protease activated receptor-1,-2,-3 and -4 in trachea isolated from influenza A virus-infected mice.

Relaxant and contractile effects of the tethered ligand domain sequences of murine PAR-1, PAR-2, PAR-3 and PAR-4, and of the proteases thrombin and trypsin were examined in mouse isolated tracheal preparations. The epithelium- and cyclo-oxygenase-dependence of these effects and the potential modulatory effects of respiratory tract viral infection were also investigated. In carbachol-contracted preparations, trypsin, thrombin, and the tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced transient, relaxant responses that were abolished by the cyclo-oxygenase inhibitor indomethacin. Repeated administration of SFFLRN-NH(2), SLIGRL-NH(2) or GYPGKF-NH(2) (30 microM) was associated with markedly diminished relaxation responses (homologous desensitization), although there was no evidence of cross-desensitization between these peptides. The tethered ligand domain sequences for PAR-1 and PAR-4 induced a rapid, transient contractile response that preceded the relaxant response. Contractions were not inhibited by indomethacin and were not induced by either thrombin or trypsin. Influenza A virus infection did not significantly affect the responses induced by either the proteases or peptides. Furthermore, epithelial disruption caused by mechanical rubbing had no significant effect on responses to these PAR activators in preparations from either virus- or sham-infected mice. In summary, the proteases trypsin and thrombin, and peptide activators of PAR-1, PAR-2 and PAR-4 induced relaxant responses of mouse isolated tracheal smooth muscle preparations, which were mediated by a prostanoid, probably PGE(2). Interestingly, PAR-mediated relaxations were not significantly diminished following acute damage to the epithelium caused by mechanical rubbing and/or the respiratory tract viral pathogen, influenza A. British Journal of Pharmacology (2000) 129, 63 - 70.

Role of PGE(2) in protease-activated receptor-1, -2 and -4 mediated relaxation in the mouse isolated trachea.

1. The potential mediator role of the prostanoid PGE(2) in airway smooth muscle relaxations induced by peptidic and proteolytic activators of PAR-1, PAR-2, PAR-3 and PAR-4 was investigated in carbachol-precontracted mouse isolated tracheal segments. 2. The tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced smooth muscle relaxation that was abolished by the non-selective cyclo-oxygenase (COX) inhibitor, indomethacin. The relative order for mean peak relaxation was SLIGRL-NH(2)>GYPGKF-NH(2) approximately amp; SFFLRN-NH(2)>SFNGGP-NH(2). 3. SFFLRN-NH(2), SLIGRL-NH(2) and GYPGKF-NH(2), but not SFNGGP-NH(2), induced significant PGE(2) release that was abolished by indomethacin. Like that for relaxation, the relative order for mean PGE(2) release was SLIGRL-NH(2)>GYPGKF-NH(2)>SFFLRN-NH(2)>SFNGGP-NH(2). 4. In dose-response studies, SLIGRL-NH(2) induced concentration-dependent increases in PGE(2) release (EC(50)=20.4 microM) and smooth muscle relaxation (EC(50)=15.8 microM). 5. The selective COX-2 inhibitor, nimesulide, but not the COX-1 inhibitor valeryl salicylate, significantly attenuated SLIGRL-NH(2)-induced smooth muscle relaxation and PGE(2) release. 6. Exogenously applied PGE(2) induced potent smooth muscle relaxation (EC(50)=60.3 nM) that was inhibited by the mixed DP/EP(1)/EP(2) prostanoid receptor antagonist, AH6809. SLIGRL-NH(2)-induced relaxation was also significantly inhibited by AH6809. 7. In summary, the results of this study strongly suggest that PAR-mediated relaxation in murine tracheal smooth muscle is dependent on the generation of the spasmolytic prostanoid, PGE(2). PAR-stimulated PGE(2) release appears to be generated preferentially by COX-2 rather than COX-1, and induces relaxation via activation of the EP(2) receptor.

Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium.

1. House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. 3. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. 4. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.

Oncostatin M synergises with house dust mite proteases to induce the production of PGE(2) from cultured lung epithelial cells.

The release of PGE(2) and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL-1beta to stimulate PGE(2) release. However, whether OSM interacts with pro-inflammatory cytokines and proteases in the production of anti-inflammatory eicosanoids and NO from airway epithelium is unknown. The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE(2) and NO production by the respiratory epithelial cell line, A549 in response to pro-inflammatory cytokines as well as protease-rich house dust mite (HDM) fractions and a protease-deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme-immunoassay. Cells treated with a mixture of IL-1beta, IFNgamma and LPS for 48 h produced a 9 fold increase in PGE(2) and a 3 fold increase in NO levels (both P<0.05). Both OSM and LIF were without effect. However, OSM added together with the cytokine mixture synergistically enhanced PGE(2) production (22 fold, P<0.05). OSM also synergistically enhanced PGE(2) production in response to a cysteine protease-enriched, but not serine protease-enriched HDM fraction (P<0.05). Rye grass extract, neither alone nor in combination with OSM, induced PGE(2) or NO production, although it did induce the release of GM-CSF. These observations suggest that OSM is an important co-factor in the release of PGE(2) and NO from respiratory epithelial cells and may play a role in defense against exogenous proteases such as those derived from HDM.

Endogenous function and biological significance of aeroallergens: an update.

Significant advances have been made in delineating the structure and function of the clinically important aeroallergens. Most have now been characterized at the molecular level, and their endogenous function determined. In the period of review, however, several novel allergens have been identified. They include the house dust mite lipophorins and gelsolins, and the birch isoflavone reductase. In addition, the functions of previously described allergens have now been established or inferred on the basis of homology studies. For example, cat Fel d 1 and the grass pollen group 1 allergens possess proteolytic activity and the thaumatin-like plant and pollen allergens possess endo-beta1,3-glucanase activity. Similarly, the lipocalin allergens may possess endonuclease activity, and the mite group 2 allergens may bind cholesterol. The three-dimensional structure of the horse dander lipocalin Equ c 1 and the honey bee Api m 2 allergens have also been determined during the review period. Finally, in this period, a variety of novel or improved immunotherapeutic allergen reagents designed to redirect the host immune response from a T-helper-2 to a T-helper-1 cell phenotype have been described, in particular, allergen and immunostimulatory CpG motif conjugates.

Partial characterisation of a soluble haemagglutinin from human diarrhoeal isolates of Aeromonas.

A soluble haemagglutinin has been identified in cell-free culture supernates of human diarrhoeal isolates of Aeromonas sobria, A. hydrophila and A. caviae. It was oligomeric; a major peak of haemagglutinating activity had an apparent mol. wt of 780,000 but there was haemagglutinating activity throughout the mol. wt range less than 40,000- greater than 10(6). Human group O, A and B, horse, rabbit, chicken and rat erythrocytes, but not those of sheep and cow, were agglutinated by the soluble haemagglutinin, in contrast to the cell-bound agglutinin. Agglutination was inhibited by fetuin, a complex glycoprotein, but not by simple sugars. The haemagglutinating activity was not affected by 0.5 M NaCl, dithiothreitol or the presence or absence of Ca++. It was unrelated to the haemolytic, enterotoxigenic and proteolytic activities present in cell-free extracts of A. sobria. All A. sobria, 73% of A. hydrophila and 68% of A. caviae strains tested produced this soluble haemagglutinin. A. caviae does not appear to be an enteric pathogen, therefore this soluble haemagglutinin alone is unlikely to be a virulence factor in Aeromonas spp.