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Monika Grote - One of the best experts on this subject based on the ideXlab platform.
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abortive pollen germination a mechanism of allergen release in birch alder and hazel revealed by Immunogold Electron Microscopy
The Journal of Allergy and Clinical Immunology, 2003Co-Authors: Monika Grote, Rudolf Valenta, Rudolf ReicheltAbstract:Abstract Background: Pollen from early-flowering trees (eg, birch, alder, hazel) represent major seasonal allergen sources. The effects of rain on the release of allergens from tree pollen has thus far not been studied at the ultrastructural level. Objective: This study was designed to investigate the effects of rain on the morphology of pollens from early-flowering trees and of potential rain-induced mechanisms of allergen release. Methods: Freshly collected pollen grains (birch, alder, and hazel) were exposed under controlled conditions to rainwater. Changes of pollen morphology and the release of allergens were analyzed by scanning Electron Microscopy. The release of allergen-bearing submicronic particles was studied by field emission scanning Electron Microscopy and transmission Electron Microscopy in conjunction with Immunogold staining by using antibodies with specificity for the major allergens. Results: Scanning Electron Microscopy showed that freshly isolated pollen grains from birch, alder, and hazel have abortive germination in rainwater. Abortive pollen germination is characterized by the formation of short pollen tubes, which rupture at their tips and release micronic and submicronic particles containing major allergens. Immunogold transmission Electron Microscopy provided evidence that the allergens are transported through the pollen tubes during germination. Conclusions: Rainwater-induced release of allergen-bearing submicronic particles from abortively germinated tree pollens may represent a mechanism of allergen release, with important implications on the induction of asthma as well as on current methods for measuring environmental allergen exposure. (J Allergy Clin Immunol 2003;111:1017-23.)
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Identification of an allergen related to Phl p 4, a major timothy grass pollen allergen, in pollens, vegetables, and fruits by Immunogold Electron Microscopy.
Biological chemistry, 2002Co-Authors: Monika Grote, Sabine Stumvoll, Rudolf Reichelt, Jonas Lidholm, Valenta RudolfAbstract:Group 4 grass pollen allergens represent 60 kDa glycoproteins recognized by 70% of patients sensitive to these pollens. An antiserum against purified Phl p 4 from timothy grass pollen was used to investigate various pollens, fruits, and vegetables for Phl p 4-related allergens by Immunogold Electron Microscopy. In timothy grass, mugwort, and birch pollens, allergens were located in the wall, and in timothy grass and birch pollens additionally in the cytoplasm. In peanut, apple, celery root, and carrot root, only cytoplasmic areas were labeled. Group 4-related allergens thus occur in pollens of unrelated plants and in plant food and may therefore contribute to crossreactivities in patients allergic to various pollens and plant food.
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in situ localization of pollen allergens by Immunogold Electron Microscopy allergens at unexpected sites
International Archives of Allergy and Immunology, 1999Co-Authors: Monika GroteAbstract:The windborne pollen grains of many trees and grasses contain a number of highly water-soluble (glyco)proteins which upon moistening, e.g. on the human respiratory mucosa, rapidly diffuse out of the pollen grain. In susceptible individuals, they may cause allergic reactions. Because of their rapid release from the pollen, pollen allergens were expected to be located in the outermost layers of the pollen grain, i.e. on the surface and in the wall (exine, intine). First attempts to localize allergens by microscopic methods in the pollen grain supported this view. However, since conventional preparation methods were used, artificial mobilization of allergens could not be excluded. Based on new, completely anhydrous preparation protocols and on the Immunogold-labeling technique, pollen allergens were Electron-microscopically shown to be located in the interior of the pollen grain, i.e. in the cytoplasm, often within ribosome-rich areas. This unexpected result was obtained in all strictly anhydrously prepared pollen species. The biological function of these allergens is largely unknown, however, clues as to possible cellular functions have been obtained for the birch pollen major allergen Bet v 1.
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monitoring of two allergens bet v i and profilin in dry and rehydrated birch pollen by Immunogold Electron Microscopy and immunoblotting
Journal of Histochemistry and Cytochemistry, 1993Co-Authors: Monika Grote, Susanne Vrtala, Rudolf ValentaAbstract: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.
Kuroiwa T - One of the best experts on this subject based on the ideXlab platform.
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Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells of Pelargonium zonale during pollen development
protoplasma, 1997Co-Authors: Nagata N, Saito C, Sakai A, Kuroiwa H, Kuroiwa TAbstract:In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen of Pelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence Microscopy after staining with 4',6-diamidino-2-phenylindole (DAPI) and by Immunogold Electron Microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by Immunogold Electron Microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells of P. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of prastids in P. zonale.Plant SciencesCell BiologySCI(E)16ARTICLE3-4217-22919
T Kuroiwa - One of the best experts on this subject based on the ideXlab platform.
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preferential degradation of plastid dna with preservation of mitochondrial dna in the sperm cells ofpelargonium zonale during pollen development
Protoplasma, 1997Co-Authors: Noriko Nagata, Chieko Saito, Atsushi Sakai, Haruko Kuroiwa, T KuroiwaAbstract:In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence Microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) and by Immunogold Electron Microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by Immunogold Electron Microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.
Peter J Peters - One of the best experts on this subject based on the ideXlab platform.
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cryo Immunogold Electron Microscopy for prions toward identification of a conversion site
The Journal of Neuroscience, 2008Co-Authors: Susan F Godsave, Holger Wille, Pekka Kujala, Diane Latawiec, Stephen J Dearmond, Ana Serban, Stanley B Prusiner, Peter J PetersAbstract:Prion diseases are caused by accumulation of an abnormally folded isoform (PrPSc) of the cellular prion protein (PrPC). The subcellular distribution of PrPSc and the site of its formation in brain are still unclear. We performed quantitative cryo-Immunogold Electron Microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrPC and PrPSc; and F4–31, which only detects PrPC in undenatured sections. At a late subclinical stage of prion infection, both PrPC and PrPSc were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrPSc may be oligomeric, protease-sensitive PrPSc.
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22 subcellular localization of rab17 by cryo Immunogold Electron Microscopy in epithelial cells grown on polycarbonate filters
Methods in Enzymology, 2001Co-Authors: Peter J Peters, Walter HunzikerAbstract:This chapter discusses the subcellular localization of Rab17 by cryo-Immunogold Electron Microscopy in epithelial cells grown on polycarbonate filters. High-resolution cryo-Immunogold EM is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Fixed material is embedded in gelatin, cryosectioned, mounted on Formvar-coated grids, and labeled. A typical labeling protocol is described based on the work by Slot and Geuze during which the sectioned material is exposed first to antibody, then to protein A-gold particles. If the primary antibody does not bind to protein A, incubation with a secondary antibody that does bind protein A can be added. The precise reaction conditions used for Immunogold labeling may vary depending on the antibody and are determined by trial and error.
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cryo Immunogold Electron Microscopy
Current protocols in pharmacology, 1999Co-Authors: Peter J Peters, Erik Bos, Alexander GriekspoorAbstract:This unit describes subcellular localization of proteins/antigens using high-resolution cryo-Immunogold Electron Microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Support protocols describe how to embed fixed material in gelatin, cryosection, and mount the sections on Formvar-coated grids. This unit is accompanied by eleven videos that illustrate many of the procedures used in this unit.
Haruko Kuroiwa - One of the best experts on this subject based on the ideXlab platform.
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improved procedure for Immunogold Electron Microscopy rapid freeze substitution with absolute acetone
Cytologia, 1997Co-Authors: Hidenori Takahashi, Haruko Kuroiwa, Shinya Miyagishima, Kyoko Toda, Ryuuichi Itoh, Tsuneyoshi KuroiwaAbstract:We investigated the best fixation methods for the Immunogold Electron Microscopy. Rapidfreeze substitution is better than conventional chemical fixation for preservation of structural integrity of cell nucleus, mitochondrion and plastid. When samples were embedded in LR White resin, fixation in absolute acetone gave a 2.6-fold increase in immuno-labelling of DNA compared to fixation with 1% glutaraldehyde in acetone. In contrast, no gold particles were identified when cells were fixed by 1% OsO4 and embedded in Spurr's resin. These results indicate that the rapidfreeze substitution in absolute acetone and embedding in LR White resin is the optimal approach for Immunogold labelling.
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preferential degradation of plastid dna with preservation of mitochondrial dna in the sperm cells ofpelargonium zonale during pollen development
Protoplasma, 1997Co-Authors: Noriko Nagata, Chieko Saito, Atsushi Sakai, Haruko Kuroiwa, T KuroiwaAbstract:In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence Microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) and by Immunogold Electron Microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by Immunogold Electron Microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.
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high localization of ribulose 1 5 bisphosphate carboxulase oxygenase in the pyrenoids of chlamydomonas reinhardtii chlorophyta as revealed by cryofixation and Immunogold Electron Microscopy
Journal of Phycology, 1997Co-Authors: Eiko Morita, Haruko Kuroiwa, Tsuneyoshi Kuroiwa, Hisayoshi NozakiAbstract:The distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii Dangeard was examined using cryotechnique and conventional fixation for Immunogold Electron Microscopy. Both methods provided essentially identical results, although somewhat higher densities of gold particles indicating Rubisco molecules were recognized in the pyrenoids of cryofixed cells. The gold particles were highly concentrated in the pyrenoid matrix within the chloroplasts. Even when considering the vast difference in volume between the pyrenoid and the rest of the Chloroplast, more than 99% of the total Rubisco labeling in the chloroplast was calculated to be present in the pyrenoid matrix. High localization of Rubisco in the pyrenoid matrix was also recognized regardless of cell age, based on immunofluorescence Microscopy of the same en bloc samples. These results are inconsistent with a recent immunocytochemical study employing cryotechnique in which more than 90% of the total Rubisco was recognized in the thylakoid region (thylakoid membranes and stroma) of C. reinhardtii cells. Rubisco highly localized in the pyrenoid matrix may take part in active photosynthetic CO2 fixation and/or the CO2 concentrating mechanism.