Juan A. Seoane-Camba

Effects of air pollution on Cup a 3 allergen in Cupressus arizonica pollen grains

BACKGROUND Cupressaceae is a family of plants resistant to airborne contamination, and its pollen is the main cause of winter allergic respiratory diseases, especially in North America, Japan, and Mediterranean countries. Recently, a major allergen from Cupressus arizonica pollen grains, Cup a 3, was cloned and expressed. OBJECTIVE To study the effects of air pollution on the expression of Cup a 3, a thaumatinlike protein, in C. arizonica pollen grains using a combination of transmission electron microscopy and immunocytochemical techniques. METHODS Observations were made in mature and hydrated C. arizonica pollen grains from various regions in Spain with different degrees of air pollution. Specimens were fixed using freezing protocols, and ultrathin sections were incubated with anti-rCup a 3 rabbit polyclonal antibodies. RESULTS Labeling of Cup a 3 was detected in mature and hydrated C. arizonica pollen grains. It was more intense in pollen from polluted air regions, and abundant gold particles were observed as they were released through the pollen grain walls. Furthermore, gold particles remained abundant in the pollen cytoplasm. The labeling was noticeably lower in pollen grains from unpolluted air regions. CONCLUSIONS Cup a 3 is present in the cytoplasm and walls of cypress pollen grains during the air dispersion and hydration stages. The abundance of Cup a 3 in pollen grains under polluted air conditions indicates that these cypresses intensify their activity as a defense from environmental pollution, thus strengthening their allergenicity.

Correlation between Olea europaea and Parietaria judaica pollen counts and quantification of their major allergens Ole e 1 and Par j 1-Par j 2

BACKGROUND In patients with pollinosis, allergic symptoms are often correlated with the number of airborne pollen grains, although this correlation is not always close. The direct measurement of the concentration of aeroallergens has only recently been introduced and is an important advance in public health information systems. OBJECTIVE To compare specific quantification of aeroallergens Ole e 1 and Par j 1-Par j 2 Olea and Urticaceae pollen counts. METHODS The Hirst method sampler and the Burkard Cyclone sampler were used for pollen count and allergen quantification, respectively. The aerosol was extracted and quantified for Ole e 1 and Par j 1-Par j 2 content using enzyme-linked immunosorbent assay procedures. RESULTS Day-to-day variations were observed in both the pollen count and the amount of allergens. Pollen counts and aeroallergen quantification were closely correlated with 99% significance (Olea/Ole e 1: R = 0.892, P < .001; Urticaceae/Par j 1-Par j 2: R = 0.734, P < .001). CONCLUSION The technique for the sampling and quantification of aeroallergens presented in this article, based on enzyme-linked immunosorbent assay and applied to the protein extracts directly obtained from the bioaerosol, represents an important advance in the epidemiologic study of allergic respiratory diseases.

Pollen grain and Ubisch body development in Platanus acerifolia

Abstract Different stages of development of pollen wall, aperture and Ubisch bodies of Platanus acerifolia (Aiton) Willdenow, have been examined with transmission electron microscopy. Different histochemical techniques for glycoprotein, unsaturated lipids and neutral and acid polysaccharides have been used. At the post-meiotic stage a polysaccharide primexine or glycocalyx is formed around the young microspores. The lipidic, glycoprotein and polysaccharide pro-Ubisch bodies are located between the tapetum plasmalemma and glycocalyx. At the liberation of microspores, glycocalyx increases in conjunction with the pro-columella and the pro-foot layer; elements of the discontinuous proectexine are located in the apertural membrane; pro-sporopollenin-like material is located on the pro-Ubisch bodies. At a more advanced microspore stage, the glycocalyx is fibrillar and the pro-endexine layer is formed under a white line; the pro-Ubisch bodies show a strong lipidic and scanty glycoprotein and polysaccharide central body; the glycocalyx envelope is formed by a thin neutral polysaccharide layer. At the vacuolate stage, a fibrillar polysaccharide glycocalyx is observed on the tapetum and microspore, the ectexine and endexine are consolidated and the Ubisch bodies have the same pattern as the reticulate and spinulate exine. In ripe pollen, the intine is located, the glycocalyx is faintly contrasted and the veil of lucent pollenkitt is present on the exine. At this stage the staining for lipids, glycoprotein, acid and neutral polysaccharide in the central part of the Ubisch bodies is still marked.

Allergenic and antigenic proteins released in the apertural sporoderm during the activation process in grass pollen grains

Abstract A combination of transmission electron microscopy with immunocytochemical methods was used to localize antigenic and allergenic proteins during the maturation and activation processes of Poaceae pollen grains. The intine undergoes a series of modifications that play a decisive role in these processes. Allergenic and antigenic proteins were detected particularly in the intine of activated in vitro grass pollen grains. Labelling of antigenic proteins was more abundant and less specific than that of allergenic proteins. At the time of hydration, the operculum was lifted up, the intine was swollen and labelling of allergenic proteins appeared highly localized in the Zwischenkörper. No significant labelling was observed when the Zwischenkörper gelatinized. Immunolocalization of allergenic proteins in the activated Zwischenkörper indicated the presence of proteins related to activation of the pollen grains. This confirms that the intine function is involved in the processes of pollen tube formation and fertilization, and also suggests the possible mechanism activated in the pollen grains when allergenic proteins reach the mucosa of sensitive subjects.

An Ultrastructural Study of Pollen Grains Consumed by Larvae of Osmia Bees (Hymenoptera, Megachilidae)

Abstract Pollen samples from provisions and faeces found in the nests of four Osmia species were analyzed by SEM and TEM. The pollen grains studied were Cistus, Sonchus type oleraceus. Primus type dulcis, and Quercus type ilex. The apertures of Cistus and Sonchus pollen stored in the provisions were slightly expanded, and the cytoplasm protruded through them. Conversely, Prunus and Quercus pollen grains showed hardly any signs of such apertural protrusions. Further, the cytoplasm of Cistus and Sonchus pollen (which have thin intines) was almost entirely lacking in the pollen grains recovered from faeces, while in the faecal pollen grains of Prunus and Quercus (with thick intines) the cytoplasm was much less modified. These preliminary results indicate that both the protrusion of the cytoplasm in the provisions and the thickness of the intine may play an important role in the digestion of pollen grains by Osmia bee larvae.

Pectin distribution pattern in the apertural intine of Euphorbia peplus L. (Euphorbiaceae) pollen

Abstract. The apertural inner layer (intine) of Euphorbia L. pollen grains has a characteristic but original structure that has paired thickenings, one on either side of the colpus. To determine the nature and role of this intine layer, pollen grains of Euphorbia peplus L. were germinated in vivo and in vitro. The germination process involves wall changes that facilitate formation of the pollen tube and its subsequent growth. In the thickenings of the intine of E. peplus, the unesterified pectin epitopes are more densely localised in the inner part of the middle intine. No such epitopes are located in the intine portion adjacent to the plasma membrane (cellulosic endintine). Unesterified pectin epitopes are also localised in the outer part of the intine but are restricted to the centre of the aperture, around and in the pore. The de-esterification of pectins is very advanced at the time of dehiscence and pollen germination. The stratification of the aperture intine may take the following pathway at the time of germination: the thin outer zone of the intine in the pore region becomes disorganised and undergoes dissolution with liberation of unesterified and esterified pectins; the middle intine thickenings undergo an important elastic modification, but without liberation of unesterified pectins; the cellulosic inner intine is the progenitor of the pollen tube wall. This special intine of E. peplus is an adaptation to the hydration process preceding germination, increasing intine and pollen grain wall elasticity.

Taxonomic significance of sporoderm structure in pollen of Euphorbiaceae: Tribes Plukenetieae and Euphorbieae

A pollen sporoderm study of tribe Plukenetieae - subtribe Tragiinae and tribe Euphorbieae was carried out. Most of the species of subtribe Tragiinae examined have a reduced exine, with a perforate tectum, short and irregular columellae, and a thin, undulated or absent foot layer. The endexine is homogeneous, fissured or spongy. The intine is well developed, thicker than the exine, and usually three-layered. In the apertural region both ectexine and endexine are present but are fragmented and disorganised. Only in the clavate-baculate pollen of Tragia volubilis is the exine well-developed and the intine thinner than the exine. The thicker intine is correlated with a thin and weakly developed exine that may provide support, compensating for the severe reduction of the foot layer and the poorly developed, fissured and spongy endexine. The pollen morphology of tribe Euphorbieae is concordant with the common pollen type in the genus Euphorbia . Nevertheless the pollen grains of this tribe have a particular apertural system, that includes modifications to the ectexine, endexine and intine at different aperture levels. The most important observation was the unusual intine with two thickenings that run along each side of the aperture. In general, pollen of the Euphorbiaceae has a thick and well-developed intine. This intine may be uniformly thickened around the grain or may form a lens-shaped structure (oncus) beneath the apertures. Intine thickenings such as those described for tribe Euphorbieae are unknown elsewhere in the family. The more specialised apertural structure, described for tribe Euphorbieae, supports the circumscription of this tribe.

The role of the intine and cytoplasm in the activation and germination processes of Poaceae pollen grains

Ultrastructural modifications of the intine and cytoplasm, during the maturation, activation and germination processes are described for several Poaceae pollen grains. Allergenic and antigenic proteins were found in the non apertural intine during the times of activation and germination, using TEM immunolabelling. This fact may be related to the function of the non apertural intine during the processes of pollen activation and pollen tube formation prior to fecundation. Changes in the granular particles of the cytoplasm are described and their role in pollen wall development is suggested. The pectic‐cellulosic and callosic layers of the pollen tube were formed on the degraded intine, and a relationship between pollen tube wall development and the substances expelled from the fibrillar particles was observed. The immunolabelling of the starch granules may be in agreement with their role in the allergenic process.

Immunocytochemical localization of allergenic proteins from mature to activated Zygophyllum fabago L. (Zygophyllaceae) pollen grains.

Zygophyllum fabago L. (Zygophyllaceae) can be found in the Middle East, in North Africa and in the arid zones of the Mediterranean region. It easily establishes itself in new regions, and is considered an invasive plant. They undergo ambophilous pollination, as there is a relationship between this type of pollination and its allergenic incidence. A combination of transmission electron microscopy with immunocytochemical methods was used to localize allergenic proteins during hydration and activation processes. Germination was induced in vitro for 1,2,4,6, and 30 min. The activated proteins reacting with antibodies present in human sera from allergenic patients are found in the cytoplasm, intine, exine and exudates from the pollen grains. The activation time plays an important role on the labelling intensity. Labelling of allergenic proteins was abundant at 1 and 2 min of activation, and decreased at 4 and 6 min. The rapid activation and release of the allergenic proteins appears to be the main cause of allergenic activity of Z. fabago pollen grains.

Lipid transfer proteins in Parietaria judaica L. pollen grains: immunocytochemical localization and function

Parietariajudaica L. (Urticaceae) pollen is considered one of the most common causes of allergic respiratory symptoms in the Mediterranean area. The localization of lipid transfer proteins (LTPs) in P. judaica mature and hydrated-activated pollen grains was investigated applying a combination of transmission electron microscopy (TEM) with immunocytochemical methods. Our results show that the content of LTPs in P. judaica pollen grains changes during the process of hydration. The localization of judaica LTPs in the cytoplasm and in the lipid bodies associated with vacuoles demonstrated that LTPs represent primarily intracellular proteins. On the other hand, exposure of the pollen grains to germination medium induced the release of LTPs from the pollen grain. Thus, LTPs are cytoplasmic proteins that are secreted to become available for pollen-stigma interactions and probably induce the IgE antibody responses in allergic patients.