Search PubMed⌕ Search

Biomedical subjects

F Ferreira

Publications and source records attributed to F Ferreira.

At least 91 records · Page 5Linked to original sources
PubMed1995

Structural studies of the extracellular polysaccharide from Butyrivibrio fibrisolvens strain 49.

The structure of Butyrivibrio fibrisolvens strain 49 capsular polysaccharide has been investigated mainly by sugar and methylation analysis, partial chemical degradations, NMR spectroscopy, and mass spectrometry. The results suggest that the polysaccharide is composed of pentasaccharide repeating units having the following structure. [formula: see text] The polysaccharide contains O-acetyl groups, one of which is substituted to O-3 of the 4-substituted alpha-D-Galp residue, while others occur in non-stoichiometric amounts at other locations.

Bacterial Capsules↗
PubMed1995

Isoforms of Bet v 1, the major birch pollen allergen, analyzed by liquid chromatography, mass spectrometry, and cDNA cloning.

Bet v 1, the major allergen of birch pollen, displays a considerable degree of heterogeneity. Several charge variants have been detected by two-dimensional IgE immunoblots and isoelectric focusing techniques. This heterogeneity has been attributed to glycosylation (or other post-translational modifications) or to isogenes coding for Bet v 1 isoforms and/or allelic variants. However, until now, only limited structural data for Bet v 1 have been published. Recently, we described the expression, purification, and immunological properties of recombinant Bet v 1 (rBet v 1) produced in Escherichia coli as a non-fusion protein (Ferreira, F. D., Hoffmann-Sommergruber, K., Breiteneder, H., Pettenburger, K., Ebner, C., Sommergruber, W., Steiner, R., Bohle, B., Sperr, W. R., Valent, P., Kungl, A. J., Breitenbach, M., Kraft, D., and Scheiner, O. (1993) J. Biol. Chem. 268, 19574-19580). Here, we present a more detailed structural characterization of Bet v 1 by both cDNA cloning and mass spectrometry. Thirteen different cDNA clones coding for Bet v 1 isoforms were obtained by polymerase chain reaction amplification of birch pollen cDNA with a sequence-specific 5'-terminal primer and a nonspecific 3'-terminal primer or by immunological screening of a birch pollen cDNA library. These isoforms are referred to as Bet v 1b to Bet v 1n, whereas the previously isolated Bet v 1 cDNA (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935-1938) is now referred to as Bet v 1a. High performance liquid chromatography and plasma desorption mass spectrometry of proteolytic fragments of purified natural Bet v 1 (nBet v 1) and rBet v 1a were used to (i) confirm the primary structure of all Bet v 1 isoforms and (ii) to investigate any possible postsynthetic modifications on rBet v 1a or on the natural mixture of isoallergens obtained from birch pollen. Except for the cleavage of initiating methionine, no postsynthetic modifications were found in either nBet v 1 or rBet v 1a.

Allergens↗
PubMed1994

Four recombinant isoforms of Cor a 1, the major allergen of hazel pollen, show different reactivities with allergen-specific T-lymphocyte clones.

Purified preparations of allergenic proteins from plants, and in particular from pollens, consist of multiple closely related isoforms. These isoforms are highly similar in their amino acid sequences, yet they display different properties with respect to antibody binding. In this study we report of differential potencies of cross-reacting tree pollen allergens and cloned isoforms of these allergens to activate allergen-specific T-lymphocyte clones (T-cell clones; TCC). Six TCC with specificity for Bet v 1, a representative tree pollen major allergen, were established from peripheral blood of five birch-pollen-allergic donors. All TCC displayed the helper-cell phenotype. Five TCC reacted with distinct epitopes present on natural (n) and on recombinant (r) Bet v 1. One TCC could not be stimulated with r Bet v 1, in spite of strong reactivity with purified natural Bet v 1. The TCC were tested in proliferation assays using purified n Bet v 1, n Cor a 1 (the homologous major allergen of hazel pollen), r Bet v 1, four recombinant isoforms of Cor a 1 and peptides representing corresponding T-cell stimulating regions (isoepitopes) on these proteins. The clones showed different patterns of reactivity in response to stimulation with the five recombinant molecules and the corresponding peptides. Certain exchanges of amino acids within stimulating peptides correlated with a lack of proliferation of the TCC tested. These findings are important with respect to the use of broadly cross-reactive recombinant allergens or allergen-derived peptides for immunotherapy of type I allergy.

Allergens↗
PubMed1994

Cloning, sequencing and transcriptional studies of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120.

In some cyanobacteria and eukaryotic algae, cytochrome c-553 (c-552) and plastocyanin function as alternative electron carriers between the cytochrome b6-f complex and Photosystem I. In these organisms plastocyanin is the electron carrier under copper-replete conditions, and cytochrome c-553 is the electron carrier during copper deprivation. In this paper we report the cloning, sequencing and transcriptional analysis of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120. The gene for cytochrome c-553 encodes a preprotein containing 111 amino acids with a predicted N-terminal transit peptide sequence of 25 amino acids. The gene for plastocyanin encodes a preprotein containing 139 amino acids with a N-terminal transit peptide sequence of 34 amino acids. RNA transcript analyses indicate that the expression of the genes for cytochrome c-553 (petJ) and plastocyanin (petE) are regulated in reciprocal ways in response to copper concentration. In copper-replete conditions, petJ is expressed at very low levels, but is transcribed at high levels under copper deprivation; petE is down-regulated in the absence of copper, but is rapidly up-regulated when copper is added back to the medium.

Amino Acid Sequence↗
PubMed1994

Molecular and functional characterization of allergens: basic and practical aspects.

Well-defined allergen preparations are, in the first line, a prerequisite for exact diagnosis, but will be supposedly useful tools in immunotherapy of Type I (IgE-mediated) allergic diseases. The allergens have to be available in standardized and highly purified form in sufficient quantities. By applying recombinant DNA techniques this goal can be achieved with respect to both, characterization and reproducibility of allergen preparations. As an example, purified recombinant non-fusion Betv1 revealed identical immunological properties with respect to interaction with both, anti-Betv1 antibodies and Betv1-specific T cell clones when compared with natural Betv1 purified from birch pollen. Moreover, cloning of allergens yielded a number of deduced primary structures of allergens, which allows computer-aided comparisons with already known amino acid sequences. Significant sequence similarities with well-described proteins may point at a biological and biochemical function of the cloned allergen, which might be of interest for considerations why a certain protein within an extract represents an allergen. Furthermore, the interaction of small peptides synthesized according to amino acid sequences of cloned allergens with allergen-specific T and B cells can be investigated. Respective results will yield information about the regulation of IgE synthesis and, thus, might point at new concepts of immunotherapy.

Allergens↗
PubMed1994

B-cell epitopes of allergens determined by recombinant techniques; use for diagnosis and therapy of type I allergy.

In the present manuscript, the immunological and functional in vitro properties of recombinant plant allergens are summarized. Recombinant tree pollen allergens (major birch pollen allergen-BetvI, birch profilin-BetvII) and recombinant timothy grass pollen allergens (PhlpI, PhlpV, and PhlpII) were compared with the natural counterparts regarding IgE-binding properties and capacity to release histamine from patients' basophils. In addition, experimental in vivo models of Type I allergy, based on recombinant allergens, are discussed. The major conclusion is that recombinant allergens can be seriously considered as candidates for diagnosis of Type I allergy allowing to establish specific allergograms for the individual patients. The in vivo data obtained in mouse and primate systems indicate that recombinant allergens can be used to set up close-to-man models of Type I allergy. Such in vivo models are useful to test the effects of already established therapeutic approaches and also allow to develop therapeutical concepts which are based on the use of recombinant allergens. Examples of specific therapeutical concepts are presented.

Allergens↗
PubMed1993

Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen--Cas s 1.

Pollens from trees of the order Fagales (e.g. birch, alder, hazel, and hornbeam) all contain one major allergen--the main cause for tree pollen allergy. So far the major allergens from birch (Bet v 1), alder (Aln g 1), hazel (Cor a 1), and hornbeam (Car b 1) have been characterized, showing high sequence similarity with each other (1-4). We present the molecular and immunologic characterization of Cas s 1, the major allergen from the European chestnut (Castanea sativa). From aqueous pollen extracts from European chestnut a protein was purified to homogeneity and was subjected to further investigation. The protein revealed a Mr of 22 kDa and was shown to represent the major allergen of the European chestnut (immunoblotting, histamine release) and designated Cas s 1. Despite a marked difference in Mr, Cas s 1 shows significant amino acid sequence similarity at the N-terminus and is antigenically closely related to the major birch pollen allergen Bet v 1 (17 kDa), as shown by binding to the anti-Bet v 1 monoclonal antibody BIP-1 and by IgE-inhibition tests using recombinant Bet v 1.

Allergens↗
PubMed1993

Identification of profilin as an actin-binding protein in higher plants.

Profilin is a low molecular weight protein involved in the organization of the mammalian and protozoan cytoskeleton as well as in signal transduction. In this study, profilin is identified as an actin-binding protein in higher plants which is present in monocot and dicot angiosperms. Birch pollen profilin and actin can be copurified as a complex, and purified recombinant birch profilin can be used as an affinity matrix to obtain birch pollen actin. The binding of 125I-labeled recombinant birch pollen profilin to plant and animal actins can be blocked by profilin-specific antibodies that react with different epitopes of birch profilin. One of the blocking antibodies was raised against the 25 COOH-terminal amino acids indicating the importance of this region in the profilactin complex formation.

Actins↗
PubMed1993

Four recombinant isoforms of Cor a I, the major allergen of hazel pollen, show different IgE-binding properties.

Previous studies showed that pollens from trees of the order Fagales (e.g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross-reactive between these tree species, and approximately 95% of tree-pollen-allergic patients display IgE binding to these allergens. Using the reported N-terminal amino acid sequence of the hazel pollen allergen Cor a I, it was possible to amplify Cor-a-I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3'-non-coding regions. Within the overlapping regions, the nucleotide sequence of the 3'-non-coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor a I. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor a I isoforms revealed identities of 96-99%. The sequence identities between the Cor a I isoforms and Bet v I, the major birch pollen allergen, were 71-73% (80.5-83% similarity). Comparing amino acid sequences of Cor a I isoforms with the published sequences of Aln g I, the major allergen from alder, and Car b I and isoforms, the major allergen from hornbeam, 75.5-76.7% identity (83.6-85% similarity) and 83.6-89.9% sequence identity (89.3-95% similarity), respectively, was found. The four Cor a I cDNAs were subcloned into plasmid pKK223-3 and expressed in Escherichia coli as non-fusion proteins; their capacity to bind serum IgE from tree-pollen-allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen-derived Cor a I. IgE antibodies from tree-pollen-allergic patients reacted with all four recombinant isoforms. However, we noted marked differences in the IgE-binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti-(Bet v I) mAb, BIP 1. Our results demonstrate that Cor a I isoforms display different antigenic and allergenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies.

Allergens↗
PubMed1993

Identification of multiple T cell epitopes on Bet v I, the major birch pollen allergen, using specific T cell clones and overlapping peptides.

Eleven T cell clones (TCC) with specificity for Bet v I were established from the peripheral blood of six birch pollen allergic donors. Bet v I is the major allergen of birch (Betula verrucosa) pollen and shows high homology to the major allergens of pollens of other trees within the order fagales (hazel, alder, hornbeam, oak, etc.), which represent important inhalant allergens in the northern hemisphere. The TCC were shown to react with purified natural, as well as with purified recombinant Bet v I. All clones showed the helper cell phenotype (CD3+CD4+) and expressed the TCR-alpha/beta. The cytokine production pattern in response to stimulation with allergen resulted in enhanced production of IL-4 in 9 of 11 clones. The clones were used for T cell epitope mapping on the Bet v I molecule. For this purpose, peptides with a length of 12 amino acids each and overlapping for 10 residues were synthesized following the amino acid sequence of Bet v I. These 75 peptides were used to stimulate Bet v I-specific T cell clones. Our experiments revealed 7 distinct T cell epitopes on the Bet v I molecule. The epitopes were scattered over the whole molecule, 2 sequences were in agreement with an algorithm previously described for the prediction of T cell epitopes. In 3 cases, we could identify distinct TCC specificities within single individuals. Furthermore, for each donor, none of the peptides representing epitopes for TCC inhibited the binding of IgE antibodies to Bet v I. These results suggest that T cells and IgE antibodies from the same individual recognize different structures on the Bet v I allergen.

Allergens↗
PubMed1993

Multiple T cell specificities for Bet v I, the major birch pollen allergen, within single individuals. Studies using specific T cell clones and overlapping peptides.

Twenty-five T cell clones specific for Bet v I were established from the peripheral blood of two birch pollen-allergic patients. The T cell epitopes of these clones were mapped using dodecapeptides overlapping for 2 amino acids (neighbors share 10 residues) spanning the whole amino acid sequence of the protein (159 amino acids). In total, 7 epitopes could be detected. One donor displayed 6 distinct T cell specificities for the Bet v I molecule in 14 T cell clones; for the other donor, 4 stimulating peptides for 11 clones could be identified. Two T cell epitopes were recognized by both subjects. One of these might represent an immunodominant epitope located at amino acid position 77-92 of the Bet v I molecule, as in 13/25 T cell clones activation could be induced by this amino acid sequence. One T cell clone reacted with purified pollen-derived Bet v I, but neither with any peptide synthesized according to a Bet v I-encoding cDNA nor with the respective recombinant protein. Upon stimulation with allergen, the majority of the clones (21/24) revealed the TH0 or TH2 type of cytokine production (interleukin-4 production), indicating their importance in the pathogenesis of the allergic disease.

Allergens↗
PubMed1993

Induction of specific histamine release from basophils with purified natural and recombinant birch pollen allergens.

As much as 15% of the population in industrialized countries suffers from type I allergic symptoms (rhinitis, conjunctivitis, and bronchial asthma). One approach toward this disease involves the production of recombinant allergens in Escherichia coli and their purification for diagnostic and therapeutic purposes. In this study we compared the IgE-binding capacity of natural and recombinant birth allergens with their functional ability to release histamine from allergic patients' basophils via cross-linking of high-affinity Fc epsilon-receptors. The recombinant as well as pollen-derived Bet v I and birch profilin (Bet v II) were purified and tested in parallel on IgE immunoblots and in in vitro histamine release tests (n = 21). We observed an excellent correlation between allergen-induced histamine release and birch pollen RAST (r = 0.881, p < 0.002). Nonspecific histamine release as a result of a cell toxic effect of the allergen preparations was never observed from basophils of donors without birch pollen allergy. The specificity of the presented effector model is also documented by the specific desensitization of patients' basophils with the recombinant allergens. These data may provide a basis for the use of purified recombinant allergens in sensitive and specific in vitro allergy tests monitoring the effector situation in allergic patients, which therefore may represent a possible alternative for in vivo diagnostic methods.

Allergens↗
PubMed1993

T cell clones specific for Bet v I, the major birch pollen allergen, crossreact with the major allergens of hazel, Cor a I, and alder, Aln g I.

Tree pollens are responsible for type I allergies during the flowering season in spring. Pollens from birch, hazel and alder constitute the most important allergen sources in this respect in the northern hemisphere. Human IgE antibodies, specific for the major allergens of these pollens, are known to crossreact, and in general every tree pollen allergic patient is sensitized to these three pollen allergens. In this study we investigated eight T-helper cell clones (CD3+, CD4+, TCR alpha/beta) with specificity for Bet v I, the major birch pollen allergen, as proved by reactivity with purified natural as well as with recombinant allergen. The T cell clones were used to investigate common T cell epitopes of the Bet v I molecule with Cor a I, the major allergen of hazel pollen and Aln g I, the major allergen of alder pollen. All eight T cell clones reacted with all three proteins with different intensity. Moreover, three T cell clones, which were known to react with immunodominant T cell epitopes on the Bet v I molecule, were tested for reactivity with dodecapeptides synthesized according to the corresponding homologous regions of the Cor a I and Aln g I sequence. All the peptides induced strong T cell proliferation, indicating the existence of multiple cross-reacting epitopes. These findings will have an impact on the production of vaccines for immunotherapy of tree pollen allergies.

Allergens↗