Flexible enzymatic activation of artificial polyketide extender units by Streptomyces cinnamonensis into the monensin biosynthetic pathway.

Streptomyces cinnamonensis A495 is a variant of the monensin producer which instead of the native polyether antibiotic gives rise to antibiotic and anti-tumour shunt-product premonensin. Through the supplementation of the fermentation medium with suitable precursors, premonensin can be derivatized via the incorporation of new-to-nature extender units into the biosynthetic machinery. Polyketide extender units require activation, typically in form of coenzyme A-thioesters. These are membrane impermeable and thus in the past an artificial mimic was employed. Here, we show the use and preliminary characterization of a highly substrate promiscuous new enzyme for the endogenous thioester formation in a Streptomyces strain. These intracellularly activated alternative extender units are significantly better incorporated into premonensin than the synthetically activated counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyketide natural products are of enormous relevance in medicine. The hit-rate in finding active compounds for the potential treatment of various diseases among this substance family of microbial origin is high. However, most polyketides require derivatization to render them suitable for the application. Of relevance in this field is the incorporation of artificial substances into the biogenesis of polyketides, hampered by both the microbial metabolism and the complexity of the enzymes involved. This manuscript describes the straightforward and selective biosynthetic incorporation of synthetic substances into a reduced polyketide and showcases a promising new enzyme to aid this purpose.

Antibodies induced with recombinant VP1 from human rhinovirus exhibit cross-neutralisation.

Human rhinoviruses (HRVs) are the major cause of the common cold and account for 30-50% of all acute respiratory illnesses. Although HRV infections are usually harmless and invade only the upper respiratory tract, several studies demonstrate that HRV is involved in the exacerbation of asthma. VP1 is one of the surface-exposed proteins of the viral capsid that is important for the binding of rhinoviruses to the corresponding receptors on human cells. Here we investigated its potential usefulness for vaccination against the common cold. We expressed VP1 proteins from two distantly related HRV strains, HRV89 and HRV14, in Escherichia coli. Mice and rabbits were immunised with the purified recombinant proteins. The induced antibodies reacted with natural VP1 and with whole virus particles as shown by immunoblotting and immunogold electron microscopy. They exhibited strong cross-neutralising activity for different HRV strains. Therefore, recombinant VP1 may be considered a candidate HRV vaccine to prevent HRV-induced asthma exacerbations.

Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. METHODS: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. RESULTS: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an alpha-helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X-ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients' IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti-Der p 21 IgG antibodies inhibited mite-allergic patients' IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross-inhibition studies identified it as a new mite allergen. CONCLUSIONS: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.

Constrained disequilibrium values and hitchhiking in a three-locus system.

Positive selection on a new mutant allele can increase the frequencies of closely linked alleles (through hitchhiking), as well as create linkage disequilibrium between them. Because this disequilibrium is induced by the selected allele, one may be able to identify loci under selection by measuring the influence of a candidate locus on pairwise disequilibrium values at nearby loci. The constrained disequilibrium values (CDV) method approaches this problem by examining differences in pairwise disequilibrium values, which have been normalized for two- and three-locus systems, respectively. We have investigated in detail the reliability of inferences based on CDV, using simulation and analytical methods. Our main results are (i) in some circumstances, CDV may not distinguish well between a selected locus and a neighboring neutral locus, but (ii) CDV seldom indicates "selection" in neutral haplotypes with moderate to large 4Nc. We conclude that, although the CDV method does not appear to precisely locate selected alleles, it can be used to screen for regions in which hitchhiking is a plausible hypothesis. We present a microsatellite data set from human chromosome 6, in which constrained disequilibrium values suggest the action of selection in a region containing the human leukocyte antigen (HLA)-A and myelin oligodendrocyte glycoprotein (MOG) loci. The connection between hitchhiking and disequilibrium has received relatively little attention, so our investigation presents opportunities to address more general issues.

Molecular characterization of an autoallergen, Hom s 1, identified by serum IgE from atopic dermatitis patients.

Atopy is a genetically determined disorder that affects 10%-20% of the population. Many symptoms of patients with atopy (allergic rhinitis, conjunctivitis, asthma, and anaphylaxis) result from events occurring after crosslinking of cell-bound IgE by per se innocuous environmental antigens. The frequently raised hypothesis that autosensitization can also be a pathogenetic factor in atopy, gained support by our recent demonstration of IgE antibodies against human proteins in atopic dermatitis patients. To unravel the molecular nature of IgE-defined autoantigens, we used serum IgE from atopic dermatitis patients to screen a human epithelial cDNA expression library. One of the cDNA-encoding IgE-reactive products contained 1501 bp of a 2274 bp open-reading frame finally identified by sequence analysis of two additional cDNA clones resulting from oligonucleotide screening. The IgE-defined autoantigen, designated Hom s 1, exhibited an almost complete sequence identity with a recently described antigen recognized by cytotoxic T cells of a squamous cell carcinoma patient. Purified recombinant Hom s 1 specifically bound IgE from patients with severe atopy. When used as immunogen in rabbits, recombinant Hom s 1 gave rise to an anti-serum that reacted with a cytoplasmic protein exhibiting a broad cellular and tissue reactivity (skin, lung >> gastrointestinal tract >> muscle, brain) and identified a 55 kDa protein in blotted serum IgE preparations. The attractive possibility remains that the Hom s 1-triggered IgE response contributes to the events resulting in allergic tissue inflammation. If so, the respective recombinant molecule may serve as a paradigmatic tool for the diagnosis and treatment of patients with "intrinsic" atopy.

pET-prof, a plasmid for high-level expression of recombinant peptides fused to a birch profilin-derived hexadecapeptide tag: a system for the detection and presentation of recombinant antigens.

We have previously identified a birch pollen profilin hexadecapeptide (Bp36/51), which was recognized by a monoclonal antibody (moAb 4A6) with high affinity. Here, we report the construction of a T7 RNA polymerase-driven high-level plasmid expression system, pET-prof, capable of producing proteins and peptides containing the Bp36/51 birch profilin-derived peptide fused to their N-terminus. As examples, the cDNAs coding for two major timothy grass (Phleum pratense) pollen allergens, Phl p 2 and Phl p 6, as well as for an alder (Alnus glutinosa) pollen allergen, Aln g 4, were overexpressed in Escherichia coli as BP36/51-tagged proteins. All three recombinant allergens were readily detected in nitrocellulose-blotted E. coli extracts by the Bp36/51-specific moAb 4A6. We demonstrate comparable IgE recognition of Bp36/51-tagged and untagged recombinant allergens by immunoblotting. A sandwich ELISA was developed using plate-bound moAb 4A6 to immobilize and present Bp36/51-tagged recombinant allergens to IgE antibodies of allergic patients. Using immunoelectronmicroscopy, we demonstrate that even under harsh fixation conditions, tagged allergens can be localized simultaneously in situ by moAb 4A6 and allergen-specific antisera. We suggest the use of the pET-prof system for the high-level expression of Bp36/51-tagged polypeptides that can be rapidly detected in total protein extracts, immunolocalized in situ, immobilized and presented to other antigen-specific antibodies (e.g. IgE), even when they occur in minute concentrations.

Immunogold electron microscopy of soluble proteins: localization of Bet v I major allergen in ultra-thin sections of birch pollen after anhydrous fixation techniques.

To localize the highly water-soluble major allergen Bet v I in ultra-thin sections of birch pollen, pollen grains were cracked, air-dried, and processed for electron microscopy using one of the following preparation techniques: fixation in aqueous p-formaldehyde + cetylpyridinium chloride; fixation in p-formaldehyde vapor; fixation in benzoquinone vapor; inert dehydration; or no fixation. Afterwards the pollen grains were embedded in Lowicryl K4M resin at low temperature. Ultra-thin sections were cut and incubated with a monoclonal antibody against Bet v I, followed by a gold-labeled secondary antibody. In some experiments, commercial rabbit IgG antibodies against birth pollen allergens were also used, followed by incubation with the protein A-gold complex. Bet v I could be localized only after vapor fixation and in the inert dehydrated specimens. Best preservation of ultrastructure and antigenicity was obtained after p-formaldehyde vapor fixation. Bet v I antibody binding sites were detected only in the cytoplasmic matrix of the pollen grain, never in the pollen wall. Commercial rabbit antibodies bound to cytoplasm and wall of all prepared specimens, even after aqueous fixation. This might be explained by the assumption that these antibodies recognize a variety of antigenic and allergenic structures, not all of which are so highly soluble as Bet v I.

Techniques to preserve soluble surface components in birch pollen wall: a scanning and transmission electron microscopic study.

The exine of birch pollen was examined by scanning and transmission electron microscopy in the native state and after fixation in different aqueous fixatives: glutaraldehyde + OsO4; glutaraldehyde + cetylpyridinium chloride (CPC) + OsO4; glutaraldehyde + cuprolinic blue (CB); and periodate + lysine + paraformaldehyde (PLP). The native pollen exine showed a thin (3-5-nm) border of electron-dense material lining the tectum and electron-dense material within microchannels and bacula cavities. Fixation with the addition of CPC resulted in a voluminous surface coat surrounding the pollen grain, but empty microchannels and bacula cavities. After fixation with the addition of CB, there was a thin surface coat, whereas microchannels and bacula cavities were partially filled with electron-dense material. The other fixatives led to empty microchannels and bacula cavities. There was no surface coat on the pollen grain. However, after all fixation procedures, a thin electron-dense border of the tectum remained visible. Concerning the electron-dense material filling microchannels and bacula cavities in the native pollen grain, the results obtained in the present study suggest that it is either completely lost (after conventional and PLP fixation) or, after fixation with a precipitating additive, partially (CB) or completely (CPC) solubilized and precipitated on the surface of the pollen grain as a surface coat.

Cross-reactivity of birch anthers and leaves with birch pollen antigens and allergens. A fine-structural immunocytochemical study using the post-embedding protein-A-gold technique.

Ultra-thin sections of vegetative tissues from birch (anthers and leaves) were labeled for pollen antigens and allergens using a commercial rabbit IgG antibody preparation directed against birch pollen antigens and allergens. Antibody binding sites were visualized using the protein A-gold technique. Specific labeling occurred in anther tissue (tape-tum cells, anther wall cells) as well as in the birch leaf (assimilation parenchyma). In both types of tissue, antigens and allergens were detected throughout the living protoplast (including cell organelles such as nuclei, mitochondria, and plastids). The cellulose cell walls were always free from anti-birch-pollen IgG-binding sites. The immunological controls (normal rabbit IgG) showed a low degree of nonspecific labeling. In plant tissues belonging to genera quite different from birch (tulip anther, rhododendron leaves), after incubation with the specific IgG weak labeling was observed. The immunological basis for these results is discussed.

Monitoring of two allergens, Bet v I and profilin, in dry and rehydrated birch pollen by immunogold electron microscopy and immunoblotting.

Dry and rehydrated birch pollen grains were anhydrously fixed and double immunogold-labeled for the presence of two allergens, Bet v I major allergen (17 KD) and profilin (14 KD). In dry pollen grains, both allergens are found exclusively inside the cytoplasm. In pollen grains rehydrated for 1 min, the cytoplasm is partially devoid of the two allergens, whereas the pollen wall and the germination aperture are specifically labeled. Pollen grains rehydrated for 5 min are largely free of the two allergens. In immunoblot experiments, both allergens could be detected in the aqueous supernatants of rehydrated pollen samples within 5 min. The results obtained by both methods show the high solubility of both proteins. This makes them readily available to the immune system and characterizes them as potent allergens. Moreover, the solubilization of profilin might indicate a dissociation of the profilin-actin complex at the very first stage of pollen germination, which could favor formation of the cytoskeleton and pollen tube growth.

Electron microscopic localization of concanavalin A receptor sites in pollen surface material after fixation with glutaraldehyde-cetylpyridinium chloride.

Pollen from birch trees (Betula pendula) was fixed in glutaraldehyde containing 0.5% cetylpyridinium chloride (CPC), incubated with concanavalin A (Con A)-ferritin, postfixed in osmium, dehydrated, and embedded in Epon. On ultrathin sections, ferritin particles were observed closely associated with the electron-dense material precipitated by CPC on the surface of the pollen grains. Controls for CPC, which were fixed in glutaraldehyde alone, showed no electron-dense material on the surface. In controls for Con A, which were incubated in Con A-ferritin in the presence of the inhibitory sugar (alpha-methyl-D-mannopyranoside), no ferritin particles were observed. The above-described procedure thus allows the localization of sugar residues in highly soluble pollen wall glycoproteins.

Immunogold electron microscopic localization of timothy grass (Phleum pratense) pollen major allergens Phl p I and Phl p V after anhydrous fixation in acrolein vapor.

We used the vapor phase of acrolein as an anhydrous fixative for timothy grass pollen in an immunogold double-labeling localization study of two different major allergens, Phl p I and Phl p V. More than 48 hr of fixation were needed for the subcellular pollen structures to be satisfactorily stabilized. The immunoreactivity of acrolein-fixed pollen allergens was not destroyed even after prolonged acrolein fixation. By immunoblotting, the two allergens differ in their immunological and structural characteristics. Electron microscopic localization traced the allergens at least partially to different subcellular pollen compartments.

Binding of antibodies against birch pollen antigens/allergens to various parts of apples as studied by immuno-gold electron microscopy.

The clinically and biochemically observed correlation between birch pollen allergy and hypersensitivity to apples was investigated by immunocytochemical techniques. For this purpose, apple tissue was fixed in p-formaldehyde and embedded in Lowicryl K4M resin at -35 degrees C. Ultrathin sections were cut and successively incubated with rabbit antibodies against birch pollen antigens/allergens and protein A/gold. Specific antibody binding sites were detected throughout the apple fruit (peel, fruit flesh, seed). Control sections incubated with normal rabbit IgG antibodies and protein A/gold showed minimal background staining. It was concluded from the results of immunocytochemical labelling that apple tissue and birch pollen contain similar molecular structures which lead to the observed cross-reactions. The present immunocytochemical results confirm biochemical investigations reporting partial structural identity of antigens/allergens in birch and apple.

Impact of PAF antagonist BN 52021 (Ginkolide B) on post-ischemic graft function in clinical lung transplantation.

BACKGROUND: Platelet activating factor (PAF) is associated with ischemia/reperfusion injury (I/R) after lung transplantation. Following promising experimental results, this prospective trial investigated the potential effect of PAF antagonist BN 52021 (ginkolide B) on clinical Euro-Collins (EC)-based lung preservation. METHODS: We analyzed 8 double-lung transplant patients in each of 3 groups. In the low-dose group (LDG), donor lungs were perfused with EC containing 2 mg/kg BN 52021, whereas we used 10 mg/kg in the high-dose group (HDG) and placebo in the control group (CG). Before reperfusing the first lung, we administered intravenously 120 mg BN 52021 (LDG), 600 mg BN 52021 (HDG), or placebo (CG). Hemodynamics in terms of pulmonary arterial pressure, pulmonary vascular resistance and serial determinations of the alveolo-arterial oxygen difference (AaDO(2)) were recorded. We measured blood levels of PAF pre-operatively and post-operatively, after 10 minutes and after 3, 8, 24, 48, and 144 hours. RESULTS: Within 32 hours, we noted a tendency toward better AaDO(2) in the LDG and the HDG compared with the CG (p > 0.05). We observed a significant improvement of AaDO(2) after 3 hours (HDG, p = 0.033) and 8 hours (LDG, p = 0.024), with poorest values in the CG. The PAF concentrations were lowest in the HDG, with significant deterioration 10 minutes after reperfusion. In contrast, placebo led to higher PAF levels. We measured significantly lower PAF concentrations (HDG vs CG) at 10 minutes and at 6 days post-operatively. CONCLUSIONS: Use of high-dose PAF antagonist BN 52021 can easily be combined with clinical preservation methods and may help optimize pulmonary function with reduced PAF levels, in the early post-ischemic period.

Ultrastructural morphology and allergen detection in birch pollen after aqueous, anhydrous-liquid, and vapor fixation techniques.

In order to find a good compromise between preservation of ultrastructural morphology and retention of antigenicity, birch pollen grains were chemically fixed in aqueous p-formaldehyde or glutaraldehyde, in p-formaldehyde or glutaraldehyde dissolved in anhydrous glycerol, and in p-formaldehyde or glutaraldehyde vapor. Representative cytoplasmic areas were inspected for the preservation of ultrastructural morphology and for their capacity to bind a monoclonal antibody against Bet v I, the major birch pollen allergen. The experiments showed that cytoplasmic morphology was best preserved after vapor fixation in p-formaldehyde. This fixation also led to the highest degree of specific antibody binding.

Expression of hepatitis B virus core gene products with specific immunoreactivity for e antigen (HBeAg) in Saccharomyces cerevisiae.

The e antigen of the hepatitis B virus (HBeAg) was expressed in S. cerevisiae. Yeast-derived HBeAg exhibits high HBe antigenicity while lacking any HBc antigenicity. The production yield of HBeAg expressed in yeast is dependent on the host strains and the nature of the leader sequences used in the plasmid constructions. The recombinant antigen is not secreted into the medium, independent from the leader sequences which are used. A simple extraction procedure was developed, enabling the isolation of HBeAg from the cells without killing them. Recombinant HBeAg derived from yeast can replace plasma-derived antigen in ELISA for determining antibodies to HBeAg.

A new protocol to prepare dry plant specimens for electron microscopy and immunocytochemistry.

Samples from dry kidney bean cotyledons were fixed in acrolein vapor and embedded in Lowicryl K4M using a modified dehydration and embedding technique. The preservation of morphology as investigated by transmission electron microscopy was excellent showing desiccated cells and organelles in life-like preservation. Postembedding immunogold labeling for kidney bean purple acid phosphatase (KbPAP) showed association of kbPAP with ribosome-rich areas of the cytoplasm.

Solvent extraction of noble metals by formazans-I Comparative study on the extractability of Pt(IV), Pd(II) and Ag(I) by formazans combined with a liquid anion-exchanger.

The extraction properties of ion-pairs composed of quaternary ammonium cations and a sulphonated formazan were compared with those of an unsulphonated formazan, for various solvent media. In dichloromethane the combined system behaves as a "coloured anion-exchanger", with displacement of the sulphonated formazan, whereas in toluene Pd(II) and Ag(I) are extracted as the metal formazan chelates from aqueous medium. The rates of extraction are remarkably higher than with the simple extractants. Because of the higher stability only the simple chelating extraction systems afford satisfactory separation of Pd(II) from excess of Pt(IV) and of Ag(I) from Cu(II). The extracted metals can be stripped and the extractant regenerated.

[Element standards for quantitative X-ray microanalysis of biological specimens in the scanning transmission electron microscope]. / Eichstandards für die quantitative Röntgenmikroanalyse biologischer Proben im Rastertransmissionselektronenmikroskop.

Quantitation in biological X-ray microanalysis usually depends on the reference to one or more element standards. This paper deals with the general characteristics of such standards and gives a survey of the various methods of standard preparation reported in literature. Their advantages and disadvantages are briefly discussed. Practical experiments were carried out to prepare a potassium, calcium, and sulfur standard for X-ray microanalysis in the scanning transmission electron microscope.