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Biomedical subjects

W M Becker

Publications and source records attributed to W M Becker.

124 records · Page 7Linked to original sources

The development of microbodies and peroxisomal enzymes in greening bean leaves.

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 microm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 microm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.

Alcohol Oxidoreductases↗

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings.

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.In seedlings grown in the dark for 10 days, the reserve lipid and the glyoxylate cycle enzyme activities persist for a longer time than in the light; correlated with this, there is a continued association of the microbodies with the lipid bodies. When these dark-grown seedlings are then exposed to 51 hours of the light-dark cycle, peroxisomal marker enzymes increase rapidly in activity, and the microbodies become appressed to chloroplasts. We conclude that the characteristic association observed between glyoxysomes and lipid bodies reflects their mutual involvement in net gluconeogenesis through the conversion of fatty acids to carbohydrate, while the close spatial relationship observed between peroxisomes and chloroplasts at later stages of development reflects their mutual involvement in glycolate metabolism.Although glyoxysomal enzyme activities are dropping rapidly while peroxisomal enzyme activities are increasing rapidly during the transition period in the light, the electron microscopic evidence does not indicate that glyoxysomes are being degraded or peroxisomes are being formed. Since in the dark-grown seedlings the activities of peroxisomal enzymes remain low and do not increase as they do in the light, an opportunity is afforded to compare quantitatively any changes in numbers of microbodies per cell with the changes in activities of glyoxysomal enzymes. It is found that the magnitude of the decrease in numbers of microbodies is considerably less than that of the decrease in glyoxysomal enzyme activities between days 4 and 10. When the cotyledons are exposed to light, peroxisomal enzyme activities increase greatly, but again there is no ultrastructural evidence for the synthesis of a new population of microbodies to accommodate this increase. These results allow us to conclude that the developmental transition from glyoxysomal to peroxisomal function almost certainly does not involve the actual replacement of one population of microbodies by another. Rather, the transition probably occurs within existing particles, either by a sequential functioning of two different kinds of microbodies or by a change in enzyme complement within a single population. Our findings with both light- and dark-grown cotyledons favor the latter possibility. The cytoplasmic invaginations into microbodies seen during greening of both light-grown cotyledons and etiolated cotyledons exposed to light may be morphological manifestations of the mechanism by which the microbodies lose or gain enzymes.

Journal Article↗

Reactivities of immunoglobulin E and immunoglobulin G subclasses identified by isoelectric focusing-immunoprint in allergic patients.

Probing of IgE or IgG subclass reactivities on single allergenic components in an extract is tedious and time-consuming under native conditions. Isoelectric focusing immunoblotting combines a powerful, highly resolving native separation method with the specificity and sensitivity of immunological test methods. This method is easy and quick to perform. The potential usefulness of this method is demonstrated by eight examples of a type I or type III allergy analyzing IgE and IgG subclass reactivities and their distribution.

Allergens↗

Examination of microheterogeneity in grass pollen allergens.

The separation of timothy pollen extract by two-dimensional immunoblotting revealed microheterogeneity of the major allergens PhI p I and PhI p V. There was not only a diversity in size, 38 and 32 kDa for PhI p V and 37, 35, and 33 kDa for PhI p I, but also a separation into proteins of identical sizes but different pIs. Since former studies on the protein structure by amino acid analysis and N-terminal microsequencing did not reveal any differences, we examined other possibilities that might cause microheterogeneity. In allergens belonging to the PhI p I group, the variability in pI can be due to the carbohydrate structure and to the fact that charges are hidden in the interior of the protein, as shown by varying concentrations of urea. On the other hand we were not able to detect any such reasons for the existence of PhI p V isoallergens. Thus, we assume that they are stable conformational isomers of the proteins (allomorphism) or that there are only slight variations in the internal sequences of these proteins (polymorphism) causing distinct pIs.

Allergens↗

Implications of the grass group I allergens on the sensitization and provocation process.

Grass pollen allergens of group I are particularly important because of their high IgE prevalence and occurrence in all grass species. Four independent IgE-binding regions and one continous epitope were identified. The posttranslational modifications on the molecule increased allergenicity. Phl p 1 is a cysteine protease, as determined by specific substrates, inhibitors and consensus sequence motifs. In analogy to other allergens and/or proteases, we deduce that Phl p 1 might enhance the permeability of the epithelium, influence T helper cells to bias Th2, and increase the IgE production of plasma cells. Thus, the group I allergens seem to be the crucial components in a pollen extract which can mediate sensitization and enhance the triggering of symptoms leading to the persistence of a grass pollen allergy.

Allergens↗

Timothy grass (Phleum pratense L.) pollen as allergen carriers and initiators of an allergic response.

Contrary to indoor allergen exposure (e.g. house dust mite), there is no reliable quantitative association between pollen exposure and symptoms of allergic diseases. Therefore we studied localization and release of major allergens from timothy grass (Phleum pratense L.) pollen using different methods and pollen grain sources. Localization of major allergens Phl p 5 and Phl p 1 was visualized by field emission scanning electron microscopy after anhydrous fixation and immunogold silver staining in a three-dimensional reconstruction; Phl p 5 was found in the cytoplasm and on the exine, Phl p 1 in the intine. No allergens were found inside the starch granules. Allergen liberation from pollen grains was studied in vitro under physiological conditions (30 min, 37 degrees C) at pH 6. 0, 7.4 and 9.0. Besides total protein measurements in the supernatant, major allergens were determined by immunoblot, Phl p 5 was quantitated by ELISA. There were striking differences in total protein and major allergen release between freshly collected and commercially available grass pollen grains as well as among freshly collected pollen between rural meadows and areas near high-traffic roads. There was a significantly different release of total protein being lowest in supernatants from commercially available pollen grains (rural/traffic vs. commercial, p<0.001), and of Phl p 5 major allergen (rural>traffic>commercial, p<0.005). Therefore, allergen bioavailability seems to be an important parameter in order to establish reliable dose-response relationships for the outdoor allergen response. Pollen grains incubated in aqueous protein-free buffer solution were also found to secrete significant amounts of eicosanoids namely prostaglandin E2 and leukotriene B4. Pollen grains thus do not act only as allergen carriers but also might have important implications on early events as initiators of allergy.

Allergens↗

Molecular characterization of timothy grass pollen group V allergens.

Phl p V is the dominant allergen of timothy grass (Phleum pratense) with two isoforms having the apparent molecular weights of 38 (Phl p Va) and 32 kD (Phl p Vb) under Western blot conditions. Two-dimensional electrophoresis/immunoblotting reveals that each isoform is split into at least four isoallergens. Structural differences in the isoforms are shown by N-terminal sequencing (only 60% identity), by reaction patterns of monoclonal antibodies and, more convincingly, by enzymic degradation of purified isoforms followed by immunologic fingerprinting. These findings are confirmed by the deduced primary protein structure of cloned Phl p Va and Phl p Vb. Experiments with IgE--affinity-purified by immobilized recombinant allergens or their fragments--reveal identical epitopes and at least one different epitope between the isoforms. Furthermore, on Phl p Va we can localize different IgE-reactive epitopes at the C terminus as well as the N terminus. By probing serum from 11 patients on recombinant C- or N-terminal fragments, an individual reaction pattern was found. Testing the histamine liberation potency of the fragments, we found the N-terminal fragment of Phl p Va to be superior to that of the C-terminal fragment or the whole molecule. These results give insights into the variability of allergens, the individuality of human reaction patterns to epitopes and the alteration of allergenicity to higher or lower levels by fragmentation.

Allergens↗

Air pollution and allergy: experimental studies on modulation of allergen release from pollen by air pollutants.

The fact that allergic diseases increase in prevalence is a generally accepted and worldwide phenomenon. The causes for this increase are not known: only hypothetical concepts exist. Epidemiological studies comparing Eastern and Western European populations have shown a striking difference in the prevalence of respiratory atopic diseases, which is lower in the East. At the same time, different patterns of air pollution have been described, namely 'classical' type I, characterized by SO2 and dust prevailing in the East, and 'modern' type II, characterized by organic compounds, fine particles and ozone, which is more prominent in the West. Type II was associated in multivariate regression analysis with increased prevalence of IgE-mediated allergy. Pollen grains collected from industrial regions with high polyaromatic hydrocarbon load in West Germany, but not in East Germany, were shown to be agglomerated with airborne particles. In vitro exposure of pollen to particles indicated morphological changes and increased allergen release from the pollen. In vitro exposure of pollen to gaseous pollutants (SO2 and NO2) under different conditions of humidity resulted in SO2-induced, but not NO2-induced reduction of allergen release from pollen. It is concluded that the bioavailability of grass pollen allergens may be modulated by air pollutants, supporting the concept of an interaction between pollen and pollutants in the atmosphere outside the organism which in turn may affect allergy-relevant phenomena.

Air Pollutants↗

Characterization of Ara h 1 by two-dimensional electrophoresis immunoblot and recombinant techniques: new digestion experiments with peanuts imitating the gastrointestinal tract.

Peanut allergy belongs to the food allergies which are not associated with aeroallergens. It permanently affects children as well as adults. Peanuts can cause IgE-mediated life-threatening hypersensitivity reactions of the immediate type. The identification and characterization of peanut allergens are preconditions for answering the question how the allergens, which interact with the gastrointestinal tract, are presented to the immune system. This would be an important contribution to the clarification of the pathomechanism of food allergy. The identification and characterization of peanut allergens are performed by electrophoresis/immunoblot techniques with patient IgE, monoclonal antibodies and the lectin ConA. Ara h 1 is identified by N-terminal sequencing of the whole molecule and LysC cleavage products. Ara h 1 is a ConA-reactive 66-kD glycoprotein which consists of a variety of isoallergens and isoforms. Peanut experiments mimicking digestion in the gastrointestinal tract clearly demonstrate the releasability of peanut allergens in the mouth and the resistance of Ara h 1 to degradation under treatment with artificial gastric fluid.

Allergens↗

Sensitivity to aspirin: a new serological diagnostic method.

Certain adverse reactions to aspirin (ASA), nonsteroidal anti-inflammatory drugs (NSAIDs) and pyrazolone derivatives resemble IgE-mediated hypersensitivity. However, convincing evidence of antigen-antibody interactions or inhibition of the cyclooxygenase pathway of arachidonic acid metabolism, stimulating the production of leukotrienes (LTs) and decreasing the production of prostaglandins (PGs), has not been presented. In this study, two types of specific IgE antibodies have been found in six serum samples from eight ASA-sensitive patients with salicyloyl and O-methylsalicyloyl disks using the Radio Allergo Sorbent Test (RAST), whereas no positive result could be found with acetylsalicyloyl disks. Further investigation on the specificity of these IgE antibodies and the chemical structure of their epitopes were performed by cross-inhibition studies. The results are in favor of an IgE-dependent mechanism involved in ASA sensitivity, and suggest that determination of specific IgE antibodies would be a safe diagnostic method for ASA sensitivity in vitro.

Adult↗