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PubMed1991

Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements.

We have investigated the functional organization and properties of cis regulatory elements in the promoter regions of two genes from tomato (LAT52 and LAT59) that are preferentially and coordinately expressed during pollen maturation. Promoter deletion analysis in transgenic plants demonstrated that only minimal (less than 200 bp) promoter proximal regions are required for developmentally regulated expression in pollen and in specific cell types of the sporophyte. Cis-acting regulatory regions of these two promoters and of a third pollen-expressed promoter (LAT56) were characterized in detail using a transient expression assay. We identified two upstream activator regions in the LAT52 promoter and further showed that a 19-bp segment from one of those regions enhanced expression of the heterologous CaMV35S promoter in pollen. Similarities in sequence between crucial cis elements provide evidence that shared regulatory elements are involved in the coordinate expression of the LAT genes during microsporogenesis.

Base Sequence↗
PubMed1980

Neonatal birch-pollen contact and subsequent allergy to birch pollen.

It is shown by regression analysis that the risk of immediate hypersensitivity to birch pollen, as measured by a skin test, is positively correlated with the quantity of birch male flowers and the number of non-rainy days in the first birch flowering season (May) met in infancy. The risk is also dependent on the month of birth, with maximal risk associated with birth in February--April. The results suggest that early pollen contacts, particularly during the first 6 months of life, increase the risk of pollen allergy for a period of 20 years. We estimate that elimination of early birch pollen contacts could have reduced the prevalence of immediate hypersensitivity to birch by 28% in our age gorup 0--19 years.

Adolescent↗
PubMed1991

Group V allergens in grass pollens. I. Purification and characterization of the group V allergen from Phleum pratense pollen, Phl p V.

An allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated by a combination of copper chelate affinity chromatography and ion exchange chromatography. Phl p V binds IgE from serum of grass-sensitized donors as revealed in immunoelectrophoretic techniques and in SDS-PAGE immunoblot, and luminescence immunoassay (LIA) inhibition experiments indicate that the allergen represents a significant part of the IgE binding capacity of the extract. In immunoelectrophoresis, Phl p V is revealed as a single precipitate. However, molecular weight studies show that Phl p V consists of at least two isoforms with similar immunochemical properties, but with different molecular size. After SDS-PAGE treatment purified Phl p V is identified as two IgE-binding components, Phl p Va and Phl p Vb, with molecular weights 33 and 29 kD. After HPLC gel filtration, Phl p Va and Phl p Vb are identified in the major 30-kD eluate. After Sephadex G75 gel filtration of whole pollen extract, Phl p V is identified in fractions corresponding to molecular weights 47 and 25 kD. The 47-kD fraction corresponds to Phl p Va/Phl p Vb as seen in SDS-PAGE, while the 25-kD component presumably corresponds to a degradation product present in whole pollen extract. The NH2-terminal sequence of Phl p V, corresponding to approximately 10% of the molecule, has been determined. The sequence shows minor variations in some residues and contains besides many alanine residues also hydroxyproline; the sequence reveals no homologies to any known NH2 terminal sequence of other proteins. The amino acid composition, revealing 26 mole % alanine and no cysteine, does not show any similarities to other known amino acid compositions of allergens. From the amino acid composition determination and an immunoelectrophoretic comparison, Phl p V is estimated to represent 6% (w/w) of the whole pollen extract.

Allergens↗
PubMed1991

Group V allergens in grass pollens. II. Investigation of group V allergens in pollens from 10 grasses.

In an earlier study an allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated and physicochemically characterized. In this study Phl p V and immunochemically similar components from other grass pollens (group V allergens) have been investigated using immunoelectrophoretic techniques. To study the allergenic importance of the group V allergens, the allergenic compositions of 10 grass pollen extracts were investigated in crossed radioimmunoelectrophoresis (CRIE) using 20 sera from grass pollen-allergic donors. Group V allergens were identified using monospecific rabbit antibodies raised against Phl p V, anti-Phl p V, which react with other group V allergens usually producing dense precipitates in immunoelectrophoresis. In this way group V allergens were identified in eight extracts, and when present the precipitate corresponding to the group V allergen was the dominant IgE binding precipitate. All identified group V allergens bound IgE in at least 17 of the 20 investigated sera. Monospecific rabbit antibodies raised against the group I allergen of Lolium perenne (rye grass), anti-Lol p I, do not precipitate group V allergens, indicating that there are no immunochemical similarities between group I and group V allergens. In SDS-PAGE anti-Phl p V identifies IgE-binding components with molecular weights between 26 and 33 kD. In contrast, anti-Lol p I binds to components of slightly higher molecular weight. Apparently, the group V components are allergens that are physicochemically and immunochemically distinct from group I allergens.

Allergens↗
PubMed1997

Immunological changes during specific immunotherapy of grass pollen allergy: reduced lymphoproliferative responses to allergen and shift from TH2 to TH1 in T-cell clones specific for Phl p 1, a major grass pollen allergen.

BACKGROUND AND OBJECTIVE: The mechanisms operative in specific immunotherapy (SIT) of Type I allergy are not completely understood. In the present study we evaluated immunological changes during SIT in pollinosis. METHOD: Eight patients suffering from pollinosis (monosensitized to grass pollen) were treated with conventional SIT. All subjects had IgE specific for Phl p 1, a major allergen of timothy grass. In vitro changes in the immunological reactivity to grass pollen extract and to recombinant Phl p 1 were evaluated. Subjects were examined at three occasions: before, after 3 months and after 1 year of SIT. RESULTS: Serological analysis revealed a marked increase of grass pollen- and Phl p 1-specific IgG, titres of specific IgE did not change significantly. Lymphoproliferative responses to grass pollen extract and rPhl p 1 were reduced already after 3 months of treatment. Accordingly, the cloning efficiency for Phl p 1-specific T-cell clones (TCC) dropped markedly in all patients. The majority of allergen-specific TCC raised before SIT revealed a TH2-like pattern of cytokine production, TCC established after SIT revealed TH1 characteristics. This shift was due to a decrease in IL-4 rather than an increase in IFN-production by T cells. Investigations of the epitopes recognized by T cells before and after SIT did not reveal the outgrowth of new ('protecting') specificities. We could not observe induction of allergen-specific CD8+ lymphocytes (supressor cells). CONCLUSION: Our data indicate that -- on the level of TH lymphocytes -- SIT induces tolerance to the allergen and a modulation of the cytokine pattern produced in response to allergen stimulation.

Adult↗
PubMed2006

Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B.

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.

Actin Cytoskeleton↗
PubMed1984

Comparative studies on tree pollen allergens. VIII. Immunological properties of the alder (Alnus incana) pollen extract.

The immunological properties of the aqueous crude alder pollen extract (AI crude) and gel filtration fractions AI 3, AI 4 and AI 34 (pool of fractions AI 3 and AI 4) were examined by immuno- and radioimmuno-electrophoretic techniques, RAST titration, RAST inhibition and skin prick tests (SPT). In CIE, the AI crude extract and AI 34 displayed reference precipitate patterns consisting of 27 and 24 visible Coomassie brilliant blue stained lines, respectively. The CRIE allergogram performed by incubation with 18 individual reaginic sera detected three IgE-binding antigens characterized by different IgE-binding properties. Antigen No. 7 (Ag 7) was demonstrated to be the major IgE antibody-binding antigen of alder pollen, while Ag 1 and Ag 11 were classified as intermediate allergens. The allergens of alder pollen were located in fractions AI 3 and AI 4 of the gel filtration chromatogram. Ag 7 was present in both fractions as demonstrated by FRIE with autoradiography (FRIEWA) on the gel filtration fractions and tandem-CRIE of AI 3 and AI 4. The CRIE allergogram, RAST, RAST inhibition and SPT demonstrated fraction AI 34 to be allergenically representative of the AI crude extract both qualitatively and quantitatively. Thus, fraction AI 34 was considered an optimal purified allergen extract of alder pollen, a suitable material for further biochemical characterization and trials on purification of the allergenic reactive antigens.

Animals↗
PubMedDate not supplied

Cross-reacting allergens in tree pollen and pollen-related food allergy: implications for diagnosis of specific IgE.

BACKGROUND: A number of recombinant allergens are by now constituents of devices that can be routinely used for the detection of specific IgE. Therefore, the results of diagnostic procedures using conventional allergen extracts can be compared with those employing selected recombinant allergens. METHODS: Thirty-four sera from patients allergic to birch pollen were tested with the standard t3-CAP and rBet v 1a- and rBet v 2-CAP. cDNA was prepared by RT-PCR using primers according to the N terminus of purified allergens. Expression cDNA libraries were screened with IgE from selected patients. RESULTS: Twenty-four patients allergic to birch pollen showed the same RAST class with t3 as with rBet v 1a; 8 patients differed within 1 RAST class. In addition, 3 patients showed RAST class 3 with rBet v 2. Besides Bet v 1 and Bet v 2, 3 allergens from celery and avocado belonging to highly conserved protein families were cloned and sequenced. CONCLUSIONS: rBet v 1a can be expected to represent an excellent tool for the diagnosis of patients allergic to birch pollen in Central, Northern, and Eastern Europe. Still, a much higher number of patients has to be tested. For their high degree of conservation, further protein families have to be identified to explain cross-reactivities of birch pollen allergens other than Bet v 1 and Bet v 2 with, e.g., allergens from vegetable food.

Allergens↗
PubMed1997

Identification of a Ca2+ binding protein as a new Bermuda grass pollen allergen Cyn d 7: IgE cross-reactivity with oilseed rape pollen allergen Bra r 1.

cDNA clones encoding two isoforms of an allergen from pollen of Bermuda grass (Cynodon dactylon) have been isolated using IgE from allergic patients. Homologous transcripts are present in pollen of 15 other grasses tested. This allergen, tentatively designated as Cyn d 7, contains two calcium binding domains and shows significant sequence similarity with other Ca2+ binding pollen allergens, namely Bet v 4 from birch and Bra r 1 from oilseed rape. Approximately 10% of allergic sera tested showed IgE reactivity to this allergen. IgE cross-reactivity was observed between this allergen and Bra r 1 of oilseed rape. IgE reactivity of this allergen requires protein-bound Ca2+. Using IgE affinity-purified from the recombinant allergen to probe Western blots of pollen extracts Cyn d 7 has been identified as a 12 kDA protein.

Allergens↗
PubMed1984

Pollen-pistil interactions in interspecific crosses of Populus (sections Aigeiros and Leuce): pollen adhesion, hydration and callose responses.

Intersectional crosses between species of sections Aigeiros (Populus deltoides, P. X euramericana or P. nigra) and Leuce (P. alba or P. tremuloides) are known to be reciprocally incompatible. The site of pollen tube arrest is on the stigma surface in pollinations between pistils of section Leuce and pollen of section Aigeiros; tubes failed to penetrate the stigma surface. In reciprocal matings, pollen of section Leuce germinated and tubes penetrated the stigma and style, where arrest occurred. Rejection may be accompanied by swelling of the tube tips, and callose plug formation. In the cross between P. deltoides and P. alba a callose response was detected in the cell walls of the transmitting tissue, adjacent to the rejected pollen tubes.

Microscopy, Electron, Scanning↗
PubMedDate not supplied

Allergen cross-reactivity between proteins of the latex from Hevea brasiliensis, seeds and pollen of Ricinus communis, and pollen of Mercurialis annua, members of the Euphorbiaceae family.

Allergen cross-reactions among three strongly sensitizing Euphorbiaceae species, i.e., the rubber tree (Hevea brasiliensis), castor bean (Ricinus communis), and the Mediterranean weed Mercurialis annua were studied in Finnish patients (n = 25) allergic to natural rubber latex (NRL), but with no known exposure to castor bean or M. annua, and French patients allergic to castor bean (n = 26) or to M. annua (n = 9), but not to NRL. In immunoglobulin E (IgE)-immunoblotting, 28% of NRL-allergic patient sera recognized castor bean seed and 48% reacted to castor bean pollen proteins. Likewise, 35% of the NRL-allergic patient sera bound to M. annua pollen allergens. Nineteen percent of castor bean-allergic patients showed IgE to NRL and 8% to M. annua proteins. Sera from patients allergic to M. annua reacted in 44% to NRL, in 56% to castor bean seed, and in 78% to castor bean pollen proteins. In immunoblotting, castor bean seed extract inhibited the binding of NRL-reactive IgE to 20 kDa, 30 kDa of NRL, and 55 kDa of proteins; NRL extract, in turn, inhibited the binding of castor bean-reactive IgE to 14, 21-22, 29, and 32-34 kDa of castor bean proteins. In ELISA inhibition, NRL extract inhibited 33% of the binding of M. annua--reactive IgE of pooled sera to M. annua pollen. In conclusion, allergen cross-reactivity in vitro was observed among three botanically related Euphorbiaceae members, H. brasiliensis, R. communis, and M. annua, but the molecular specificity of the observed cross-reactions as well as their clinical significance remains to be elucidated. Allergen cross-reactivity should be taken into account in diagnostic work.

Adolescent↗
PubMed2006

[Study of recent yearly trend of increase in airborne pollen of Japanese cedar and cypress pollen].

BACKGROUND: Based on epidemiological studies of prevalence, sensitization as well as pollen survey, it is presumed that airborne Japanese cedar pollen (JCP) and cypress (JCyP) have increased progressively for past 40 years. However, because of their large yearly variations, accurate objective and scientific study is required to confirm if it is true or not. METHODS: We analyzed the time trends of JCP and JCy separately in 11 districts throughout Japan where have records of continuous past survey from 1986 to 2005, by regression analysis using net pollen count and their 3 and 5 running means. RESULTS: When significant slope of regression line (regression coefficient) is assumed as p < 0.05 and R2 (coefficient of determination) >0.4, significant increase in airborne pollen was revealed in the use of 5 point running mean (6 districts of total 11 in JCP and 5 in JCyP) but not net count or 3 point running mean because of correction of statistic error due to large yearly variations. This study suggested that our method used for analysis of a recent increase in airborne JCP and JCyP was useful and scientific.

Air Pollutants↗
PubMed1990

A double-blind trial of oral immunotherapy for Artemisia pollen asthma with evaluation of bronchial response to the pollen allergen and serum-specific IgE antibody.

Eighteen asymptomatic Artemisia pollen asthma patients with normal pulmonary function were selected for a double-blind trial of oral immunotherapy. Each patient had a positive skin test to Artemisia pollen extract and also a positive bronchial challenge response to the same extract. The patients were randomly assigned to active treatment or placebo group and received intensive oral administration of Artemisia pollen extract over a 50-day course. The nine patients who received the active treatment ingested a cumulative dose of 396,652 PNU and showed a significant decrease in serum-specific IgE antibodies (P less than .05) and a significant reduction in bronchial sensitivity to the same extract (P less than .01). The changes in these two variables correlated well (r = .8787, P less than .01). The nine patients who received the placebo showed no significant changes in serum-specific IgE or bronchial sensitivity to Artemisia pollen extract. Follow-up of two cases with the same extract showed that the reductions in serum-specific IgE as well as bronchial sensitivity induced by oral immunotherapy were maintained for 3 months.

Administration, Oral↗
PubMed1987

Vitamin D and its metabolites in the pollen of pine. Part 5: Steroid hormones in the pollen of pine species.

Unconjugated vitamin D and its metabolites were investigated in the pollen of Pinus nigra Ar. and Pinus sylvestris L. by TLC, HPLC and competitive radiochemical determination of 25-hydroxycholecalciferol (25-OHD3). It was found that vitamin D (D2, D3) was present in the pollen in amounts about 2 micrograms/10 g and 25-OHD3, 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] and 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] between 0.1 and 3 micrograms/10 g of pollen, dependent on pollen species and methods.

Chromatography, High Pressure Liquid↗
PubMed1987

Comparative studies on tree pollen allergens. XVII. Immunochemical analysis of the international standardization extracts of birch (Betula verrucosa) pollen as compared with a local partially purified extract.

Six different birch pollen extracts were analyzed by 20 laboratories for the standardization of birch (Betula verrucosa) pollen extracts used for diagnosis and specific therapy of patients with birch pollen allergy. The extracts were collected and delivered by the International Union of Immunological Societies, Allergen Standardization Subcommittee. One of the extracts, designated M, was proposed as an international standard (IS)-candidate of birch pollen extracts. The protein content of the IS candidate M was found to be 1.12 mg/mL, more than 2-fold higher than any of the other extracts analyzed. This preparation was among the extracts containing the highest number of protein components, as shown by isoelectric focusing, 28 lines, and by 11 precipitates in crossed immunoelectrophoresis. The allergenic reactivities were tested by crossed radioimmunoelectrophoresis (CRIE) and by radioallergosorbent test (RAST)-inhibition. In CRIE, the proposed IS (M) showed similar affinity for binding patients' IgE as the other extracts, as judged by the autoradiographic illustrations. Except for extract L, the values of RAST-inhibition for the rest were very similar. An IS extract should qualify for the criteria suggested for an optimal allergen preparation, containing minimal amounts of non-allergenic antigens and providing quantitatively and qualitatively all the allergenic proteins. The appropriateness of this selection seems unjustified in view of the above criteria.

Allergens↗
PubMed1982

Comparative studies on tree pollen allergens. II. Isolation of alder (Alnus incana) pollen allergens: purification and some characteristics of the major allergen pI 4.78.

The aqueous extract of alder (Alnus incana) pollen was found to consist of a protein mixture amounting to 3% of the dry pollen weight. Separation by gel permeation chromatography on an AcA-54 column resulted in 6 fractions designated chronologically to their relative elution volumes (AI 1 to AI 6). The immunological reactivity of these fractions was demonstrated in both IgE and IgG test systems. Being both immunochemically and quantitatively dominant fraction AI 4 was selected for further purification by a semipreparative isoelectric focusing column. A well-resolved and apparently homogeneous component with a pI value of 4.78 was eluted from the IEF column. The homogeneity of this fraction was demonstrated by one sharp band in thin layer IEF, one single band in HVE, one distinct sharp peak in HPLC and a symmetrical precipitate in CIE. The molecular weight was estimated to 22,500 daltons. Fraction pI 4.78 was shown to be a major allergen of alder pollen, it elicited a 1.17 histamine wheal reaction in skin prick test at a concentration of 1 mg . ml-1 and showed a significantly high RAST inhibition in all of the reaginic sera used. Fraction pI 4.78 was found to be an adequate model allergen of alder pollen well suited for further immunochemical characterization.

Allergens↗
PubMed1982

Comparative studies on tree pollen allergens. IV. Evaluation of two commercially available allergen extracts of alder (Alnus incana) and birch (Betula verrucosa) pollen.

Two commercially available extracts of alder and birch pollen usually used in clinical allergy were evaluated and correlated to two corresponding reference extracts 'S' produced at the authors' laboratory. The set of extracts produced by ALK, Denmark are referred to as 'A', while those produced by Nyco, Norway are referred to as 'N'. The results of the CIE analysis demonstrated that, upon using the same antibodies, both the morphology and the intensity of the precipitates of the different extracts differed considerably. In the alder pollen extract, the 'N' preparation contained more immunoprecipitate lines than the two others. Extract 'S' contained the least number of antigenic lines. Only two of the antigenic lines bound specific IgE in CRIE and were represented in variable concentrations in the three extracts. These two allergenic precipitation lines were demonstrated to have an immunological reaction of identity as shown by tandem-CIE and tandem-CRIE systems, respectively. In the birch pollen system variable numbers of reproducible antigenic lines of precipitation were demonstrated. Preparation 'A' contained more antigens than the two other extracts. This extract lacked a cathodic antigen with slow electrophoretic mobility represented in 'N' and 'S'. One dominant antigenic line was commonly present in the three extracts and could bind IgE antibodies in the CRIE system. A minor cathodic allergen was shown by CRIE in extracts 'S' and 'N', but not in extract 'A'. The RAST and RAST-inhibition titration experiments in all extracts showed qualitative and quantitative similarities. The amino acid compositions and the carbohydrate analyses showed that extracts 'S' and 'N' were similar while extract 'A' contained higher concentrations of certain amino acids and carbohydrates. In both alder and birch pollen, the reference extract 'S' contained lower concentrations of the nonallergenic antigens and of the low molecular weight contaminants.

Allergens↗
PubMed1994

[The evaluation of hyposensitization with sugi pollen extracts in patients with nasal allergy to Japanese sugi pollen].

We evaluated the clinical and pathophysiological efficacy of hyposensitization with Japanese sugi pollen extracts to sugi pollinosis. Twenty patients with Japanese cedar pollinosis received hyposensitization with Japanese cedar pollen extracts for one to 15 years with a mean of 9.5 years were compared with 18 patients without hyposensitization. The improvement of nasal symptoms was not found in patients with hyposensitization of sugi pollen extracts compared with patients with non-hyposensitization, but the number of mast cells in nasal scraping, nasal hypersensitivity to histamine, and specific sugi IgG4 antibody in serum was improved significantly compared with non-hyposensitization group. These findings suggest that the continuation of hyposensitization with sugi pollen extracts is recommendable as a basic treatment against seasonal nasal allergy.

Adult↗