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Purification and characterization of Pla a 1, a major allergen from Platanus acerifolia pollen (2002)

Asturias, J. A. ; Ibarrola, I. ; Bartolomé, B. ; [et al.]

Oxford, UK : Blackwell Science, Ltd

Allergy 57 (2002), S. 0

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ISSN: 1398-9995

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Medicine

Notes: Background: Plane trees, as Platanus acerifolia, are an important source of airborne allergens in cities of the United States and Western Europe. Little is known about the relevant allergens of this pollen. The aim of this study was to identify relevant allergens from P. acerifolia pollen and purify and characterize a major allergen of 18 kDa. Methods: P. acerifolia pollen extract was fractionated using ion exchange, gel filtration, and reverse-phase chromatography. Analyzes were carried out by EAST, SDS-PAGE, isoelectric focusing, immunoblotting and amino-acid sequencing. Results: An 18-kDa protein from the P. acerifolia pollen extract, which we named Pla a 1, was purified. This nonglycosylated protein had an isoelectric point value higher than 9.3 and was recognized by up to 92% of monosensitized Platanus allergic patients and 83% of polyzensitized patients. Sequencing of its N-terminal yielded an amino acid sequence which showed no homology to the known proteins in the databases. Other relevant allergens detected in monosensitized patients were proteins of 43 and 52 kDa, with immunoglobulin (Ig)E-binding prevalences of 83 and 42%, respectively. Profilin was an important allergen in polyzensitized patients. Conclusions: The most relevant allergens from the P. acerifolia pollen have been determined. A major allergen, specific of this pollen, and named Pla a 1, has been purified and characterized.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1034/j.1398-9995.2002.03406.x

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Biological characterization of glutaraldehyde-modified Parietaria judaica pollen extracts (2004)

Ibarrola, I. ; Sanz, M. L. ; Gamboa, P. M. ; [et al.]

Oxford, UK : Blackwell Science Ltd

Clinical & experimental allergy 34 (2004), S. 0

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ISSN: 1365-2222

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Medicine

Notes: Background Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts.Objective To assess an accurate methodology for standardization of chemically modified extracts.Methods GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured.Results Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found.Conclusions The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2222.2004.01859.x

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Pho d 2, a major allergen from date palm pollen, is a profilin: cloning, sequencing, and immunoglobulin E cross-reactivity with other profilins (2005)

Asturias, J. A. ; Ibarrola, I. ; Fernández, J. ; [et al.]

Oxford, UK : Blackwell Science Ltd

Clinical & experimental allergy 35 (2005), S. 0

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ISSN: 1365-2222

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Medicine

Notes: Background Up to now, some date palm pollen (DPP) allergens have been described but very few data are available about their molecular nature. The aim of this study was to identify and characterize Pho d 2, a major allergen from this pollen.Methods Sera from 25 patients allergic to DPP were analysed by immunoblotting. Purification of DPP profilin was performed by poly-l-proline affinity chromatography. Profilin-encoding cDNA from DPP was cloned by using a RT-PCR strategy and recombinant allergen was expressed as a non-fusion protein in Escherichia coli. Natural and recombinant Pho d 2 were investigated by means of enzyme allergosorbent test to compare the immunologic properties of both allergens and to analyse cross-reactivity with other profilins.Results A 14.4 kDa protein was identified as a major allergen in DPP extract. Purification, cloning, heterologous expression, and inhibition experiments identified it as profilin (Pho d 2). Pho d 2 comprises 131 amino acids and has high sequence identity with other allergenic food and pollen profilins. The prevalence of specific IgE antibody reactivity to natural Pho d 2 by ELISA was 56% and 64% by skin prick test (SPT). Pho d 2 is an important allergen as it is responsible for more than 70% of the IgE reactivity to the pollen extract. IgE directed against Pho d 2 showed a strong cross-reactivity with other profilins such as those from olive tree and grass pollens.Conclusion Pho d 2, a 14.4 kDa protein identified as profilin, is a major and relevant allergen in DPP, as confirmed by SPT and thereby may elicit clinical symptoms in sensitized patients.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2222.2005.02179.x

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The major Platanus acerifolia pollen allergen Pla a 1 has sequence homology to invertase inhibitors (2003)

Asturias, J. A. ; Ibarrola, I. ; Eraso, E. ; [et al.]

Oxford, UK : Blackwell Science Ltd

Clinical & experimental allergy 33 (2003), S. 0

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ISSN: 1365-2222

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Medicine

Notes: Background Sycamores or plane trees are an important source of airborne allergens in many cities of the United States and Western Europe. Pla a 1 has been described as a major allergen from Platanus acerifolia (London plane tree).Objective To clone and characterize the cDNA for Pla a 1 and to express the recombinant protein.Methods Pla a 1 was isolated by cationic exchange, gel filtration, and reverse-phase chromato-graphies. Pla a 1 cDNA was cloned by reverse transcription followed by polymerase chain reaction, using amino acid sequences from tryptic peptides of the allergen. The Pla a 1 encoding sequence has been subcloned into the pKN172 expression vector and expressed in Escherichia coli as a non-fusion protein. Purified recombinant protein has been tested for its IgE-binding capacity in immunoblot, immunoblot inhibition, and ELISA.Results Pla a 1 reacted with serum IgE from 35 of the 42 (83.3%) Platanus-allergic patients studied and represented 60% of the total IgE-binding capacity of the P. acerifolia pollen extract. The allergen displayed 43% sequence identity to a grape invertase inhibitor and showed a predicted secondary structure characteristic of all-alpha proteins. Serological analysis revealed that both natural and recombinant forms of Pla a 1 displayed similar IgE-binding capacity.Conclusions Pla a 1 belongs to a new class of allergens related to proteinaceous invertase inhibitors. Recombinant Pla a 1 binds IgE in vitro like its natural counterpart and, therefore, it can be useful for specific diagnosis and structural studies.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2222.2003.01707.x

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Quantification in mass units of group 1 grass allergens by a monoclonal antibody-based sandwich ELISA (2001)

Arilla, M. C. ; Ibarrola, I. ; Eraso, E. ; [et al.]

Oxford, UK : Blackwell Science, Ltd

Clinical & experimental allergy 31 (2001), S. 0

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ISSN: 1365-2222

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Medicine

Notes: Background Grass pollen extracts currently used for allergy diagnosis and immunotherapy are a complex mixture of proteins of which only a few have allergenic activity. Lol p 1 is one of the most important allergens in grass pollen extracts.Objectives To develop a two-site enzyme-linked immunosorbent assay for the quantification of Lol p 1 and other group 1 allergens from grass species, and to assess its suitability for quantifying this group of allergens.Methods Balb/c mice immunized with recombinant Lol p 1 were used for the production of monoclonal antibodies. Screening of hybridomas was performed by direct ELISA, and selected monoclonal antibodies were immobilized on ELISA plates and incubated with samples containing group 1 allergens. Bound allergens were detected by a combination of biotinylated Lol p 1-specific monoclonal antibody and peroxidase-streptavidin conjugate.Results The assay is based on three Lol p 1-specific monoclonal antibodies with different epitope specificities. The optimized ELISA measured Lol p 1 concentrations ranging from 125 to 1000 ng/mL and could quantify group 1 allergen from grass species belonging to the Pooidea subfamily. The assay does not depend on anti-sera production or availability of human sera and thus reactives can be produced in unlimited amounts.Conclusion This sensitive and specific Lol p 1 assay will be helpful both for quantifying the group 1 allergen content of Pooideae pollen extracts intended for clinical use and for studying cross-reactivities among pollen extracts.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2222.2001.01166.x

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Time-course of digestive-enzyme acclimation in the cockle Cerastoderma edule (1999)

Ibarrola, I. ; Navarro, E. ; Iglesias, J. I. P. ; [et al.]

Springer

Marine biology 135 (1999), S. 47-56

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ISSN: 1432-1793

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract We determined the temporal evolution of amylase, cellulase, laminarinase and protease in the digestive gland and crystalline style of cockles Cerastoderma edule held over 9 to 12 d in the presence and absence of food. Cockles were fed a constant diet of 1.5 mm3 l−1 of Tetraselmis suecica for 9 to 12 d and were then starved for 6 to 8 d in late summer (September 1992) and in winter (January 1993). Feeding increased the dry weight and total cellulase, laminarinase and protease activities of the digestive gland irrespective of season, whereas amylase activity remained unchanged. In winter (i.e. when cockles are metabolically weak) the response was faster and stronger, especially for protease. An additional experiment in September starved cockles for 20 d before resuming feeding. In agreement with the seasonal differences, the presence of food after prolonged starvation induced a rapid and marked increase in protease in the digestive gland of the cockles. In winter, the possible effects of the biochemical composition of food on their enzymatic response were tested by feeding two groups of cockles with the same ration of T. suecica but harvested at different growth phases. A compensatory induction of cellulases occurred in cockles fed on T. suecica with a lower carbohydrate content. In the crystalline style, the protein level and carbohydrase fell during the first day of feeding and increased during the first day of subsequent starvation. These results indicate that the release of enzymes from the style prevails over the incorporation of enzymes during the early stages of feeding, whereas the opposite occurs during starvation.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002270050600

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Short-term adaptation of digestive processes in the cockle Cerastoderma edule exposed to different food quantity and quality (1998)

Ibarrola, I. ; Navarro, E. ; Iglesias, J. I. P.

Springer

Journal of comparative physiology 168 (1998), S. 32-40

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ISSN: 1432-136X

Keywords: Key words Cockles ; Digestion ; Enzymes ; Gut capacity ; Absorption of biochemical components

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract Feeding and digestive parameters were analysed in cockles Cerastoderma edule fed for 3 days on two foods of different qualities, both foods given in two different concentrations. With low quality food, gut content was found to increase with ingestion rate. Such increased capacity of the gut to allocate food precludes negative effects upon throughput time, and so absorption efficiency remained nearly constant at the two food concentrations. With high quality food, gut content remained at high constant values and consequently enhancement of food ingestion rate with a high food ration leads to a significant reduction in throughput time, resulting in lower absorption efficiencies. Significantly higher levels of amylases and cellulases have been found within the digestive gland of cockles fed high quality diets. Coincidentally, absorption of carbohydrates is increased and absorption of lipids decreased in such diets as compared to low quality diets. Implications of the positive correlation between digestive enzyme activity and food quality are discussed in relation to the role that both digestive investments and endogenous faecal losses play in digestive processes. Results obtained in this study indicate that investments in the form of digestive enzymes are a key factor in the functional response of cockles to short-term variations in the food regime.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s003600050118

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