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A new attempt to study biomineralised silica bodies in Dactylis glomerata L.

The accumulation process of inorganic compounds in animals and plants by biomineralisation is not well understood nowadays, though it may be the key to an environmental-compatible production of modern materials in future. In this paper a new attempt will be made on the investigation of silica accumulation in grasses (especially Dactylis glomerata L.). The silicic acid agglomerates in Dactylis glomerata L. were studied by means of scanning electron microscopy in combination with energy dispersive X-ray spectrometry as well as infrared and micro-RAMAN spectrometry.In particular blades were prepared by critical point drying or shock-freezing for anatomical studies of silica cells and bristles in the plant tissue. SEM imaging and EDX microanalysis for elemental composition were done in the cryostage as well as under variable pressure. The localized silica bodies were examined for their structural properties by means of IR and micro RAMAN spectroscopy. The results are comparable to SiO(2) polytypes such as high disperse silica and opal.

Dactylis↗

Inhibition of the passive cutaneous anaphylaxis reaction to Dactylis glomerata pollen allergens by a purified component of this pollen.

By isoelectric focusing a protein fraction of pI 4.6 was isolated from a crude water-soluble extract of Dactylis glomerata pollen (SE). This fraction was neither immunogenic nor allergenic in BALB/c mice. In one week, this protein inhibited the mouse IgE-specific antibodies to the soluble extract as measured by PCA in rats and was therefore called Dactylis inhibitory protein (DIP). Two experimental approaches which lowered IgE anti-SE titer were undertaken. Pretreatment with DIP as well as injection of DIP after the last sensitizing injection with SE resulted in an inhibition of the circulating IgE antibody level to SE. For both experiments the regulation of the immune response touched only the IgE class, whereas the titers of anti-SE IgG, IgM, IgA antibodies were not modified. DIP treatment did not alter the IgE titers, measured by PCA, in the immune response to ovalbumin.

Allergens↗

Antigenic activity of nonpollen parts (leaves and stems) of allergenic plants (Parietaria judaica and Dactylis glomerata).

In patients with respiratory allergy to pollen it is common to correlate the onset, duration and intensity of clinical symptoms with the count of atmospheric allergenic pollen grains. Pollen counts, however, may not reflect the total airborne allergen exposure since previous data suggest that pollen allergens may also be carried in microaerosol suspensions. These microdroplets may penetrate deeply into the airways, where pollen grains are too large to penetrate, eventually inducing asthma. The origin of these allergenic aerosols is still uncertain. We investigated whether antigenic activity is present in vegetative parts of allergenic plants. We have used extracts from leaves and stems of Parietaria judaica and Dactylis glomerata to evaluate patients with allergic sensitization to pollen allergens of these plants (19 grass-sensitive patients and 23 Parietaria sensitive). By using skin prick testing and RAST to stem and leaf extracts other than pollen extracts we observed that most patients sensitive to grass or Parietaria pollen had small responses to extracts of stem or leaf. We conclude that allergenic components are present throughout most of Parietaria judaica and Dactylis glomerata plants, most highly concentrated in the pollen but present in the leaves with a trace in the stems.

Adolescent↗

Silica accumulation in Triticum aestivum L. and Dactylis glomerata L.

The silica accumulation in orchard grass (Dactylis glomerata L.) and wheat (Triticum aestivum L.) has been studied in plant samples grown under defined conditions in a pot trial. The plant habit and the quantity of biomineralised silica within the selected Gramineae depend to a remarkable extent on the soil. The plants grew with different soil pH values and silica additives. By means of atomic absorption spectrometry, the silicon enrichment in different plant parts was determined. In dried plant parts the silica bodies can be well distinguished by variable pressure scanning electron microscopy in the back scattering mode. They are located in silica cells below the epidermis and in epidermal appendices (bristles, prickle hairs). The silica bodies showed a defined morphology, structure and composition which was elucidated by the combined performance of scanning electron microscopy in combination with X-ray spectroscopy, solid-state nuclear resonance, X-ray diffraction and Raman spectroscopy. The composition was near to stoichiometric SiO(2) (41 weight% silicon, 56 weight % oxygen), and the SiO(4/2)tetrahedra were arranged preferentially in three-dimensional networks; a smaller proportion was in chains and layers. The silica bodies with an overall amorphous structure contained crystalline precipitates, which could be indexed by alpha-quartz.

Dactylis↗

Heptacarpo-octatarso-dactyly combined with multiple malformation.

A case of heptacarpo-octatarso-dactyly combined with cheilo-gnatho-palato-schisis, hypertelorism, macroglossia, complex malformations of heart and great vessels, horse-shoe-kidney, micro-penis, and penis palmatus is described. To our knowledge, this syndrome has not yet been described in the literature. Some of the features of the case reported overlap with syndromes such as Grauhan syndrome and Meckel syndrome.

Abnormalities, Multiple↗

Critical evaluation of an indirect enzyme-linked immunosorbent assay for IgG antibodies to Dactylis glomerata.

Experiments are reported for the evaluation of an indirect ELISA using peroxidase conjugate for the detection of IgG antibodies to Dactylis glomerata (DG), a grass pollen antigen frequently responsible for allergic rhinitis and asthma. When optimized, the test proved to be useful in clinical practice for a semiquantitative, cheap and simply-performed determination of IgG antibodies to DG. The advantages and disadvantages of this method, compared with those of elaborate antigen-binding assays, are discussed.

Allergens↗

Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata.

Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

Cell Division↗

Growth, proline accumulation and water relations of NaCl-selected and non-selected callus lines of Dactylis glomerata L.

Sodium chloride-tolerant calli were selected from leaf-derived embryogenic calli of Dactylis glomerata L. on agar solidified medium supplemented with 200 mM NaCl, a concentration lethal to non-selected calli. Growth characteristics, water relations and proline accumulation pattern were compared in selected and non-selected lines. The objective was to gain an understanding of the mechanism(s) of tolerance in the NaCl-tolerant line. Growth in the selected line, as expressed in terms of tolerance index (ratio of fresh wt. on NaCl medium:fresh wt. on NaCl free medium x 100), was greater than that of the non-selected line at all levels of NaCl between 50 and 300 mM. There was no significant difference in proline accumulation in the selected and non-selected lines. Maintenance of turgor by osmotic adjustment was observed in the non-selected line despite decreased growth. In contrast, the selected line lost either the need or the ability to adjust osmotically. There was little or no increase in symplastic osmolality in the selected line when exposed to NaCl. Presumably, selection was made for a salt-excluding tissue that has lost the ability to accumulate solutes and adjust turgor with NaCl stress.

Cell Line↗

Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen.

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

Allergens↗

Heterogeneity of grass pollen allergens (Dactylis glomerata) recognized by IgE antibodies in human patients sera by a new nitrocellulose immunoprint technique.

A new nitrocellulose immunoprint technique has been developed to detect specific antigens or/and allergens present among a heterogeneous solution such as a water-soluble crude extract of a grass pollen (Dactylis glomerata). The antigens are separated by isoelectric focusing (IEF) in an agarose gel and characterized by their isoelectric point (pI). These antigens are transferred and immobilized on a nitrocellulose sheet. They are recognized by the binding of specific antibodies contained in an unfractionated serum to be studied. Finally, the binding of these antibodies is visualized by species- or/and class-specific antibodies themselves labeled by an enzyme or by radioactivity. So one can detect the allergens recognized by the specific serum IgE antibodies and also the other antigens recognized by specific IgG, IgA or IgM antibodies.

Allergens↗

Investigations of the allergens of cocksfoot grass (Dactylis glomerata) pollen.

The pollen of cocksfoot grass (Dactylis glomerata) is an important cause of allergic reactions in man. Preliminary studies, which established that constituents of an extract of this pollen could be separated, and then recovered efficiently, by size-exclusion chromatography on TSK G3000 SW, have been extended. A comparative examination has been made by this procedure of cocksfoot pollen extracts from different sources and of several batches of extract from one source. The recovery and distribution of biological activity has been assessed by the radioallergosorbent test, and the results have been used for the selection of fractions for further investigation by chromatography and electrophoresis. Two constituents active in the radioallergosorbent test have been purified from the extracts.

Allergens↗

Characterization of Dac g 4, a major basic allergen from Dactylis glomerata pollen.

Monoclonal antibodies were produced against Dac g 4, a purified major basic allergen from Dactylis glomerata pollen. Their ability to be used for immunopurification of Dac g 4 was studied on a BIAcore apparatus (Pharmacia). The allergen was purified by affinity chromatography with one monoclonal antibody. Its precise molecular mass, 59,185 +/- 30 d, was determined by mass spectrometry. Its isoelectric point is 10.4. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting showed that Dac g 4-related proteins of similar molecular mass were detected in the majority of allergenic grass pollen species. By double-site ELISAs, we have estimated that Dac g 4 represents about 6% of the total proteins from a water-soluble extract. One monoclonal antibody (mAb H) recognized a 60 kd cross-reactive protein in other grass pollens, though none in any of the tree or weed pollens tested. Inhibition studies of IgE antibody binding to Dac g 4 with pollen extracts confirmed the presence of cross-reactive allergens in Secale cereale, Lolium perenne, Festuca elatior, Holcus lanatus, Bromus arvensis, Poa pratense, Hordeum sativum, and Phleum pratense.

Allergens↗

Production of a monoclonal antibody against a major allergen of Dactylis glomerata pollen (Dg1).

A mouse monoclonal antibody was produced against the most frequent allergen Ag Dg1ief, purified from the Dactylis glomerata grass pollen by isoelectric focusing. Using the immunoprint technique, this monoclonal antibody detected Ag Dg1ief in D. glomerata pollen and some cross-reacting molecules in four other grass pollens. This hybridoma was grown in ascitic fluid. An immunosorbent was made from it and, by affinity chromatography, was used to purify monoclonal Ag Dg1m from the pollen extract. Ag Dg1m (6,000 to 8,000 daltons) appeared to be 4 to 5 times smaller than Ag Dg1ief. It appeared as a weak allergen in passive cutaneous anaphylaxis in rats, but was more potent in triggering histamine release on human basophils from grass-pollen sensitive patients. These results suggest that Ag Dg1m could be a part of the Ag Dg1ief used to induce production of this monoclonal antibody.

Animals↗

Physicochemical and immunologic characterization of low-molecular-weight allergoids of Dactylis glomerata pollen proteins.

BACKGROUND: Orchard grass (Dactylis glomerata) pollen proteins were chemically modified by means of acid anhydrides (maleic and succinic anhydride) to obtain low-molecular-weight allergoids. Chemical modification in both cases led to the replacement of one positive charge (epsilon amino group of Lys) by one negative charge, yielding proteins with changed physicochemical properties in comparison to the native orchard grass-pollen proteins. METHODS: Physicochemical characterization of derivatives was done by gel chromatography, SDS-PAGE, and isoelectric focusing. To examine the IgE-binding properties of these derivatives, we carried out immunoblotting. To examine the ability of derivatives to induce IgG production, we immunized rabbits. Skin prick testing with the allergoids was performed on 15 individuals allergic to orchard grass pollens and on two healthy subjects. RESULTS: It was shown that the modified proteins retain their original molecular weights, but change pI to more acidic values. In the case of allergoids, a strong reduction in IgE binding was found. Immunization of rabbits with allergoids showed that the derivatives retain the ability to induce IgG production, and that the antisera obtained in such a way react to native (unmodified) extract. The ability of derivatives to induce allergic reaction was significantly reduced. The patients (86.6%) included in our study exhibited less than 50% of native extract response. Among them, 53.3% had no response to one or both allergoids. CONCLUSIONS: These modification procedures yield allergoids with a reduced allergenic activity and preserved immunogenic potential suitable for use in immunotherapy.

Allergens↗

Drought survival, summer dormancy and dehydrin accumulation in contrasting cultivars of Dactylis glomerata.

To study survival under prolonged and severe drought in the perennial grass Dactylis glomerata we compared dormant, resistant and sensitive cultivars (cvs.) in both field and glasshouse experiments. Water status, membrane stability and expression of dehydrins were assessed in the immature leaf bases, which are the last surviving organs. Analysis of leaf elongation and senescence of aerial tissues showed that dormancy was exhibited by the potentially dormant cultivar (cv.) only in the field. This cultivar exhibited a high survival rate, similar levels of dehydration and expression of a low-molecular weight (22-24 kDa) dehydrin in both drought and irrigated plants, whether fully dormant or not. At the same level of soil water deficit, there were no differences between the non-dormant drought resistant and drought sensitive cultivars in plant water status and membrane stability. However, the accumulation of dehydrins as drought progressed was markedly different between these cultivars and was associated with their contrasting survival. The possible role of the major low-molecular dehydrins in maintenance of cell integrity under dehydration is discussed with reference to both summer dormancy and survival under severe drought.

Journal Article↗

Chloroplast DNA variation in dactylis glomerata L. taxa endemic to the macaronesian islands

In the Dactylis glomerata infraspecific polyploid grass complex, restriction fragment length polymorphisms (RFLPs) of chloroplast DNA (cpDNA) were studied in diploid and tetraploid populations of several taxa endemic to Macaronesia (Madeira and the Canary islands) and in populations from the African and European continental areas closest to Macaronesia. Two chlorotypes, which differed by a single 290-bp length mutation, were observed in the Macaronesian and the continental populations. Chlorotype I, which is predominant in the whole D. glomerata complex, was found in the majority of continental populations. It was also observed in the most western Macaronesian islands, in the two diploid taxa endemic to the lowland scrub and the high elevation heath of Tenerife, respectively, and in tetraploids endemic to Madeira and La Palma. These island populations were growing under the influence of humid trade winds. Chlorotype II was found in the eastern part of the Archipelago (closer to Africa), which experienced subarid Mediterranean climate conditions, and in very few diploid and tetraploid Mediterranean populations growing at high elevation on the continent. This geographical and climatic distribution of chlorotype variation in Macaronesia is consistent with that reported previously for morphological, allozyme and phenolic variation in the same plant material. Chlorotype II was, however, also observed in tetraploid populations from La Gomera island and in one of the seven tetraploid populations analysed from Madeira, which all showed clearly subtropical characters for morphology, allozymes and phenolic compounds. This result suggests that cpDNA introgression has occurred more than once from the Mediterranean material into the subtropical one and may indicate that colonization between the mainland and islands, or among the islands, probably played a major role in the geographical pattern observed for that marker.

Journal Article↗

The effects of intake of lucerne (Medicago sativa L.) and orchard grass (Dactylis glomerata L.) hay on the motility of the forestomach and digesta flow at the abomaso-duodenal junction of the sheep.

1. The relations between food intake, reticulo-ruminal motility and abomasal digesta outflow were investigated in ewes receiving lucerne (Medicago sativa) v. orchard grass (Dactylis glomerata) hay. Abomasal digesta outflow was recorded continuously by means of an electromagnetic flowmeter probe inserted into a duodenal T-shape cannula and the motility using strain-gauge force transducers. Volumes and turnover rates of both rumen and abomasal liquid phases were measured by CrEDTA dilution. 2. The voluntary intake of lucerne was higher (60%) than that of orchard grass hay, and paralleled by an increased abomasal outflow (65%) corresponding to an increased number of gushes of digesta through the flowmeter probe: 129/h instead of 78/h on orchard grass hay. Abomasal motor activity was enhanced (35%), and periods of regular spiking activity were seen passing along the duodenum at a higher velocity for the lucerne diet than for the orchard grass diet. The increased abomasal outflow with lucerne hay was associated with a higher reticulo-ruminal turnover rate, but not abomasal turnover rate. Abomasal, but not reticulo-ruminal volume, was increased (30%) when lucerne hay was fed ad lib. 3. The total number of reticulo-ruminal contractions was increased by 6.6% when the dry matter entering the duodenum was increased by 70.6%, suggesting the level of voluntary intake, rather than reticulo-ruminal motility, as a major factor governing abomasal outflow in sheep. 4. More frequent passages of digesta, unrelated to duodenal contractions, were recorded in ewes receiving lucerne compared with orchard grass. Both a higher viscosity of the contents due to the addition of guar gum and the impairment of antroduodenal motility by 5-hydroxytryptophan were able to reduce the flow rate of orchard grass digesta, but did not affect the higher flow rate of lucerne digesta. The findings suggest that the high abomasal outflow in sheep fed on lucerne ad lib. is related to a low viscosity of the contents that are propelled, even in the case of antral contractions of low magnitude.

Action Potentials↗