Biomedical subjects

W M Becker

Publications and source records attributed to W M Becker.

At least 73 records · Page 4Linked to original sources

PubMed1992

Epitope analysis of the allergen ovalbumin (Gal d II) with monoclonal antibodies and patients' IgE.

Ovalbumin (OVA) is a major allergen (Gal d II) of hen egg white and is often the cause of hypersensitivity reactions to food. Further knowledge of the antigenic and allergenic epitopes of allergens will provide better treatment of this disease. To analyse these epitopes we produced a panel of monoclonal antibodies (mAbs) against native OVA. The initial information about the epitopes was obtained with the binding patterns of these mAbs in IEF-immunoprints and western blots of OVA under reducing and non-reducing conditions. It was possible to demonstrate that the different conformations of OVA exhibit different epitopes, and that there are other epitopes which are shared by each conformation. Seven different, although sometimes overlapping epitopes, could be determined on native OVA; four different epitopes on denaturated non-reduced OVA by means of immunoblots of the intact molecule. The number of epitopes which could be differentiated by the mAbs was increased by the use of peptide blots after CNBr fragmentation of the molecule. IgE binding to different OVA conformations and to CNBr-fragments of OVA was also detectable and appears in the same regions as the reactivity of some mAbs. Western blots of OVA and CNBr-peptides demonstrate that some antigenic/allergenic binding sites seem at least partly to be continuous epitopes. The identification of the CNBr-fragments was performed by a microsequence analysis of blotted CNBr-fragments after a 2-dimensional electrophoresis. IgE was found to bind the two largest CNBr-fragments (residues 41-172 and 301-385), but not the fragment corresponding to residues 173-196. A number of monoclonal antibodies also reacted with the two large fragments, especially with fragment 301-385, and some bind also to shorter peptides, such as fragment 173-196, which were not reactive to patients' IgE. Most of the monoclonal antibodies and patients' IgE bind to the fragments 41-172 and 301-385 in 2D-PAGE blots suggesting that these fragments are involved in an immunogenic structure.

Amino Acid Sequence↗

PubMed1992

Photooxidative Destruction of Chloroplasts Leads to Reduced Expression of Peroxisomal NADH-Dependent Hydroxypyruvate Reductase in Developing Cucumber Cotyledons.

Photooxidative destruction of chloroplasts by exposure of norflurazon-treated cucumber (Cucumis sativus L.) seedlings to white light leads to reduced levels of the nuclear-encoded, peroxisomal enzyme hydroxypyruvate reductase. The partial reduction in hydroxypyruvate reductase activity under photooxidative conditions is accompanied by reductions in levels of hydroxypyruvate reductase protein and transcript. The low level of hydroxypyruvate reductase gene expression in the dark is not affected by norflurazon, and nonphotooxidizing far-red light is able to induce significant increases in hydroxypyruvate reductase expression even in the presence of norflurazon. We conclude that intact plastids are required for maximal expression of hydroxypyruvate reductase in the light and that the plastids affect hydroxypyruvate reductase gene expression at a pretranslational level.

Journal Article↗

PubMed1992

Characterization of isoforms of the major allergen Phl p V by two-dimensional immunoblotting and microsequencing.

Timothy pollen extract was separated by 2D PAGE blot for further characterization of the major allergen Phl p V. Using pooled patient serum, we demonstrated that Phl p V consists of 4 components at 32 kD and 4 components at 38 kD, each differing in their pIs. The primary structure and the amino acid composition of the 8 proteins were determined form the blotted samples. Proteins of the same molecular weight did not differ in the 20 N-terminal amino acid residues, whereas the 32- and 38-kD proteins showed only 60% sequence identity. Furthermore, the results of the amino acid analyses suggest differences in their protein structure. Therefore, it might be possible that both proteins express different IgE-reactive epitopes.

Allergens↗

PubMed1992

Detection of IgE antibodies specific for 1-phenyl-2,3-dimethyl-3-pyrazoline-5-one by RAST: a serological diagnostic method for sensitivity to pyrazoline drugs.

Certain adverse reactions to pyrazoline drugs resemble IgE-mediated hypersensitivity. However, convincing evidence of antigen-antibody interactions is not fully demonstrated. In this study, IgE antibodies specific for 1-phenyl-2,3-dimethyl-3-pyrazoline-5-one have been found in 17 out of 19 serum samples from individuals sensitive to pyrazoline drugs with 4-aminoantipyrine discs by Radio Allergo Sorbent Test (RAST). In contrast, we have not found any positive results from 10 normal donors without sensitivity to pyrazoline drugs after ingestion of metamizol 500 mg/day for 14 days. Therefore, our results provide further evidence in favor of an IgE-dependent mechanism in patients suffering from sensitivity to pyrazoline drugs. The determination of specific IgE antibodies could be used as a serodiagnostics method.

Adult↗

PubMed1992

The presence of specific IgE to salicyloyl and O-methylsalicyloyl in aspirin-sensitive patients.

Certain adverse reactions to aspirin (ASA), nonsteroidal anti-inflammatory drugs (NSAIDs) and pyrazoline derivatives resemble IgE-mediated hypersensitivity. However, convincing evidence of antigen-antibody interactions or of the inhibition of the cyclooxygenase pathway of arachidonic acid metabolism leading to an increase in the generation of leukotrienes (LTs) and a decrease in the generation of prostaglandins (PGs) was not fully demonstrated. In this study, two types of specific IgE antibodies have been found in 27 serum samples from 28 ASA-sensitive patients with salicyloyl-discs and O-methylsalicyloyl-discs by Radio Allergo Sorbent Tests (RAST). The positive rates were 96.4% and 71.4%, respectively. In contrast, no positive results could be found in 10 normal donors without ASA sensitivity after ingestion of ASA 500 mg/day for 14 days. Further investigation of the chemical structure of epitopes was done by cross inhibition studies. Our results are an increasing evidence in favour of an IgE-dependent mechanism in patients suffering from ASA sensitivity. Hopefully, the determination of specific IgE antibodies will be a safe diagnostic method of ASA sensitivity in vitro.

Adult↗

PubMed1991

Characterization of genes encoding hydroxypyruvate reductase in cucumber.

Several clones corresponding to the gene encoding NADH-dependent hydroxypyruvate reductase have been isolated from a cucumber genomic library. Restriction mapping indicates the presence of two HPR genes, hpr-A and hpr-B, in the cucumber genome. Examination of the DNAs of individual plants suggests that hpr-A and hpr-B are most likely alleles at a single locus. The sequence of a 6.7 kb genomic fragment that includes the entire transcribed region, 2.2 kb of 5' flanking sequence, and about 0.8 kb of 3' flanking sequence reveals the presence of 12 introns in hpr-A. These introns are AT-rich relative to the exons. The donor sequence at the 5' end of the sixth intron contains an unusual dinucleotide, GC, rather than the nearly invariant GT. Primer extension analysis maps the transcription initiation site to 61 nucleotides upstream of the translation initiation codon. An AT-rich stretch is centered at position -31 with respect to the transcription initiation site, and a potential CCAAT box is centered at position -138. Several elements that are homologous to regulatory elements of other plant genes have been identified in the flanking regions of hpr-A.

Alcohol Oxidoreductases↗

PubMed1991

Differences in clinical and immunologic reactivity of patients allergic to grass pollens and to multiple-pollen species. II. Efficacy of a double-blind, placebo-controlled, specific immunotherapy with standardized extracts.

The IgE response of patients only allergic to grass pollens differs from response of patients allergic to multiple-pollen species. The IgE immunoblots to orchard-grass pollens confirmed that polysensitized patients had more proteins revealed than patients only allergic to grass pollens. To determine if both groups of patients present a different response toward specific immunotherapy (IT), a double-blind, placebo-controlled study was performed in 70 patients. Patients receiving the active treatment had a rush IT with either a standardized orchard grass-pollen extract or with a standardized mixed-pollen extract prepared, depending on the sensitivity of the patients. The maintenance dose was defined as that dose effective in grass-pollen IT in previous experiments. The same equipotent maintenance dose was administered for all pollen species. Symptom-medication scores during the pollen season and nasal challenge with orchard grass-pollen grains demonstrated that grass pollen-allergic patients had a significantly improved efficacy by comparison to placebo treatment, whereas polysensitized patients had a nonsignificant improvement. Serum grass-pollen IgG was significantly increased after IT in both treated groups. This study demonstrate that the response toward specific IT differs in patients only allergic to grass pollens by comparison to polysensitized patients.

Adolescent↗

PubMed1991

Clinical and immunologic reactivity of patients allergic to grass pollens and to multiple pollen species. I. Clinical and immunologic characteristics.

The heterogeneity of pollen-allergic individuals is well-known but poorly characterized. Twenty-six patients were studied to characterize their immunologic and clinical patterns. Thirteen patients were allergic only to grass pollens, and 13 other patients were allergic to grass-pollen and other pollen species, including Cupressaceae, plane tree, olive, and Parietaria. The IgE response was assessed by the titration of serum total IgE and orchard grass-specific IgE, as well as by IgE immunoblots to orchard-grass pollens. Clinical reactivity was assessed by nasal challenge with orchard-grass pollens before the pollen season and nasal and bronchial symptom-medication scores between April 1 and June 15. Pollen counts were obtained during this period of survey. Polysensitized patients had significantly increased levels of serum total and specific IgE and a greater heterogeneity of IgE immunoblots, suggesting an enhanced qualitative and quantitative IgE immune response. Polysensitized patients had nasal and bronchial symptoms occurring earlier than grass pollen-allergic individuals, confirming the priming effect caused by other plans flowering with an earlier season for both nasal and bronchial mucosa. Alternatively, the early symptoms may be attributable to the tree pollens or might reflect the higher grass-pollen IgE level in the polysensitized group. Bronchial symptoms appeared a few weeks after nasal symptoms. Nasal challenges were similar in both groups, and the severity of nasal symptoms during the season was not significantly different in both groups, suggesting that the intensity of symptoms is not related to the sensitization nor to the IgE immune response of the subjects.

Adolescent↗

PubMed1991

Immunological cross-reactivity of hemoglobins in the Diptera family Chironomidae.

Polyclonal human IgE antibodies of patients sensitized to one species of the Diptera family Chironomidae as well as polyclonal rabbit anti-Chi t I hyperimmune serum recognize antigenic sites present in nearly all of the 33 species of this insect family. Evolutionary distantly related genera usually show weaker antibody binding. According to the pattern of reactivity of rabbit anti-Chi t I-component III, expression of the epitopes of this molecule varies considerably in the genera Chironomus and Glyptotendipes; it appears to be almost totally absent in all other species. Of five monoclonal antibodies raised against Chi t I-component III, two recognize an epitope which is expressed in nearly all closely related species. Three monoclonal antibodies recognize epitopes which are expressed in only a few species of the same genus. Our results demonstrate the presence of common as well as species-specific epitopes in chironomid hemoglobins which behave as potent inhalant allergens.

Animals↗

PubMed1991

Release of mediators from human gastric mucosa and blood in adverse reactions to benzoate.

A study was carried out on 29 patients to investigate the amount of histamine liberation and release of platelet-activating factor and 6-keto-prostaglandin F1 alpha from gastric mucosa and whole blood or mononuclear cells by sodium benzoate. The patients suffered from asthma (10), atopic dermatitis (7) and chronic urticaria (4). 8 patients with unrelated, non-immunologic diseases served as controls. In the oral provocation test (OPT) 3 patients experienced a recurrence of their original disease, whilst 1 asthmatic patient reacted with abdominal disorder. The release of histamine and prostaglandin from mucosa was significantly increased by sodium benzoate in comparison to the spontaneous release observed in patients. The mucosa of the control persons did not react to sodium benzoate. Furthermore, there was a significant difference in prostaglandin release between patients with positive OPT and the control persons. No difference could be found between patients with negative OPT and those with positive OPT. Additionally, in the mediator release from whole blood or mononuclear cells there was no obvious difference apparent. These results suggest a possible involvement of prostacyclin and histamine in adverse reactions to benzoate. Due to the sensitivity of the method, a mediator release from mucosa can already be demonstrated in a preclinical state of the pseudoallergic reaction in the absence of clinical symptoms.

6-Ketoprostaglandin F1 alpha↗

PubMed1991

Epitope mapping of allergens and antigens of timothy pollen extract.

For the identification of allergens in crude timothy pollen extract, 2D immunoblotting was performed with subsequent use of patients' IgE. Only about 15 of more than 70 detectable components with molecular weights of 55, 38, 35, and 32 kD showed IgE-reactivity. The 55 and 35 kD allergens were identified as glycoproteins by a glycan detection kit. To investigate the protein structure of the allergens, proteins of the pollen extract were cleaved by CNBr and endoproteinases. The cleavage products were identified by patients' IgE and monoclonal antibodies (moab) raised against timothy pollen proteins. While the use of endoproteinases resulted in a complete loss of some allergens, CNBr produced smaller IgE-reactive proteins, e.g. the 38 kD component was split into a 32 kD component and the original 32 kD protein was partially cleaved into IgE-reactive fragments of 30, 21, and 15 kD. In order to investigate the IgE-binding epitopes we intend to determine the amino acid sequence of the cleavage products.

Allergens↗

PubMed1990

Organ Specificity and Light Regulation of NADH-Dependent Hydroxypyruvate Reductase Transcript Abundance.

By probing total RNA blots with an NADH-dependent hydroxypyruvate reductase (HPR) cDNA clone, we have found that HPR transcript abundance is highly regulated in developing cucumber (Cucumis sativus) seedlings. HPR transcript levels in light-grown seedlings are 50-fold more abundant in leaves and cotyledons than in roots. When 12-day light-grown seedlings are shifted to the dark for 4 days, the HPR transcript level in leaves drops 20-fold. Upon return of these dark-adapted plants to the light, HPR transcript levels rise to 50% of the previous light-grown level within 2 hours.

Journal Article↗

PubMed1990

[Exogenous allergic asthma following pectin exposure--a new occupational allergen].

In a 51-year-old male worker, workplace-related dyspnoea in the sense of bronchial asthma, was observed. On the basis of the medical history, the results of serology, inhalatory provocation and histamine release tests, despite a negative skin test, pectins were established to be the precipitating substances; in experiments on animals (Wistar rats), the instillation of pectins led to the development of bronchiolitis. An exogenous, allergic asthma following exposure to pectin may be considered to have been proven.

Air Pollutants, Occupational↗

PubMed1989

Monoclonal antibody that defines the prostate specific antigen.

A monoclonal antibody to a protein with a single band in the 30-kD region was obtained from fusion of Balb/C mouse spleen cells immunized with epithelial fractions of BPH, with the myeloma cell line P3 x 63 Ag8 6.5.3 by standard procedures. This antigen was characterized in immunobinding studies with various cellular and target antigens and in immunoperoxidase staining.

Animals↗

PubMed1989

Isolation, characterization and sequence analysis of a full-length cDNA clone encoding NADH-dependent hydroxypyruvate reductase from cucumber.

A full-length cDNA encoding NADH-dependent hydroxypyruvate reductase (HPR), a photorespiratory enzyme localized in leaf peroxisomes, was isolated from a lambda gt11 cDNA library made by reverse transcription of poly(A)+ RNA from cucumber cotyledons. In vitro transcription and translation of this clone yielded a major polypeptide which was identical in size, 43 kDA, to the product of in vitro translation of cotyledonary poly(A)+ RNA and subsequent immunoprecipitation with HPR antiserum. Escherichia coli cultures transformed with a plasmid construct containing the cDNA insert were induced to express HPR enzyme activity. RNA blot analysis showed that HPR transcript levels rise significantly in the first eight days of light-grown seedling development. This closely resembles the pattern seen for HPR-specific translatable mRNA. DNA blot analysis indicated that a single HPR gene is likely present per haploid genome. Nucleotide sequence analysis revealed an open reading frame of 1146 bases which encodes a polypeptide with a calculated molecular weight of 41.7 kDa. The derived amino acid sequence from this open reading frame is 26% identical and 50% similar to the amino acid sequence of the E. coli enzyme phosphoglycerate dehydrogenase, which catalyzes a similar reaction and functions in a related pathway. Statistical analyses show that this similarity is significant (z greater than 10). The derived amino acid sequence for HPR also contains the characteristics of an NAD-binding domain.

Alcohol Oxidoreductases↗

PubMed1989

[Production and characterization of monoclonal antibodies against allergen components].

Monoclonal antibodies are useful tools for characterizing components causing type I allergy. Three aspects in production and characterization of monoclonal antibodies are discussed: 1. production of monoclonal antibodies raised against a special allergen of rye pollen extract. 2. investigation of monoclonal antibodies raised against peptide fragments of ovalbumin, 3. analysis of epitopes on allergenic components of timothy pollen extracts. The results indicate that monoclonal antibodies raised against certain allergens are useful for isolation of allergenic components and epitope mapping.

Animals↗

PubMed1989

[Use of monoclonal antibodies in allergy research--chance of a further development?].

In diagnostics and therapy of allergic diseases preparations of natural origin are mainly used. Standardizing these products monoclonal antibodies could play an important role. Furthermore, for monoclonal antibodies there is an analytical approach measuring allergen pollution, but also the possibility to use the antibodies modified in a therapeutic way or as an aid to raise monoclonal antibodies with certain properties (e.g. affinity).

Antibodies, Monoclonal↗

PubMed1989

[Aspergillus fumigatus: evaluations of the standardization of diagnostic extracts].

Extracts of Aspergillus fumigatus used for serodiagnosis of allergic diseases are of unknown and nonstandardized composition. Applying immunoprint technique subsequent to isoelectric focusing commercial and self-prepared extracts are compared. By use of an ABPA-pool serum and monoclonal antibodies recognizing antigens of this fungus, different immunological reactivities of the extracts can be demonstrated. It is suggested to use monoclonal antibodies as secondary standards as well as to improve standardization.

Antibodies, Monoclonal↗