Academic literature on the topic 'Bermuda grass Pollen'

Pollen is responsible for seasonal allergies, such as allergic rhino-conjunctivitis (AR), and has become a growing public health concern. Climate change affects the range of allergenic species as well as the timing and length of the pollen season. In Egypt, data on pollinosis are scarce. This study aimed to identify the most prevalent pollen causing allergies among Egyptian patients with respiratory allergies. A total of 200 patients with respiratory allergic diseases, allergic rhinitis and/or bronchial asthma (BA), were included. Medical history taking and physical examinations were conducted on each patient. Complete blood count (CBC), total immunoglobulin E (IgE) determination, spirometry, specific IgE, and skin prick tests (SPTs) for common aeroallergens and food were performed. Of the 200 patients, 106 (53%) were females. The age of study subjects ranged 16-66 years (mean ± SD, 34.42 ± 13.0), and 65% were living in urban areas. Grass pollen, mainly from Timothy grass and maize, were the most prevalent allergens (28.5%). Timothy grass was the most common type of pollen in patients with AR (28.3 %). Elder pollen was more prevalent among asthmatic patients (P = 0.004). Bermuda grass was statistically more prevalent in rural than in urban areas (P = 0.008). Maize was linked to uncontrolled BA, whereas Timothy grass was the most prevalent among patients with moderate/severe AR. Forty-three patients had oral allergy syndrome; oranges and tomatoes were the most cross-reactive food allergies (12% and 11.5%, respectively). Exacerbation of allergic symptoms was noted during January, December, March, and June. In conclusion, pollen plays a substantial role in affecting patients with respiratory allergies in Egypt. Grass pollen is the most prevalent type of pollen, especially in urban areas.

碩士
國立臺灣大學
分子醫學研究所
87
BG60, having an apparent molecular weight of 60 kD, is one of the major allergens in Bermuda grass. SDS-PAGE analysis revealed that BG60 contained at least 3 components with slight differences in molecular weight (designated L and S for large and small forms respectively). The peptide sequences show that L and S are isomers. Database searching with the peptide sequences revealed high similarity to a plant reticuline oxidase (BBE), especially a putative FAD binding motif. BG60 exhibited a fluorescence emission maximum at 450 nm, and denaturation BG60 failed to eliminate the auto-fluorescence. These suggest that BG60 is a putative FAD covalent linked flavoprotein. Prediction of the secondary structure by CD suggested thatβ-sheet is the major secondary structure of BG60. Limited proteolysis suggested that L consists of an N-terminal segment, a 33 kD central domain, and a 22 kD C-terminal domain, whereas S lacks the N-terminal segment. The lower protease susceptibility, the higher melting temperature, and the preference for crystal growth of L forms than S forms implied the importance of the N-terminal segment in the structural stabilization. Green crystals of BG60 were obtained in 30% PEG4K and 25% isopropanol. The crystals diffract beyond 2 A resolution and belong to a tetragonal space group (P422) with the unit cell dimensions a = b = 86 A, c = 310 A. There are two monomers in an asymmetric unit. It still needs more efforts to find the heavy atom derivative to resolve the three dimensional structure of BG60.

博士
國立臺灣大學
生物化學暨分子生物學研究所
93
Hypersensitivity is one of the most irritating diseases. It results in atopic disorders, such as urticaria, edema, dermatitis, rhinitis and asthma. And sometimes, lethal systematic anaphylaxis and allergic shock will also occur. Among the allergic diseases, type I hypersensitivty mediated by IgE is the most acute and suffering. Here, we focused on the identification and immunologic characterization of these important allergens triggering type I hypersensitivity. First, we identified and characterized alliin lyase as the important allergen from garlic (Allium sativum). We have purified the allergenic alliin lyase, and investigated the allergen by using periodate-oxidation assay, IgE-crossreactivity assay, IgE-inhibition assay and intradermal skin test. In this study, we have demonstrated that the garlic allin lyase triggered type I hypersensitity, presented IgE-crossreactivity and IgE-inhibition with the enzymes from shallot, leek and onion, and showed weaker IgE-binding after its carbohydrates oxidized. Second, we established the 2-D protein profile and 2-D IgE-reactive profiles of Bermuda grass pollen (BGP) for an overview of allergenic proteins. By using our newly designed “major allergen depletion approach” and “multiple identification process”, an protein identification process combining of N-terminus homology, peptde-mass fingerprinting, peptide sequence identification, enzyme activity assay and specific Abs recognition, we have successfully identified six novel IgE-reactive proteins from BGP, they are enolase 2, aldolase, elongation factor 2, malate dehydrogenase, pathogenesis-related protein 1, and Phl p 11 homologues. The allergenic enolase 2 with high prevalence was formly named as Cyn d 22 and further investigated. We have cloned and purified the recombinant Cyn d 22, and also demonstrated that native Cyn d 22 could tirgger histamine release, PBMC proliferation, cytokine production, and showed IgE-crossreactivity and IgE-inhibition with enolase from S. cerevisiae. This is the first time to identify and characterize garlic and BGP allergens by a combination of proteomic and immunologic approaches. This strategy provides an efficient and useful tool for the identification and characterization of novel allergens and should greatly improve the studies in allergy sensitization. Of significance, it may also provide important information on the development of a new class of biopharmaceuticals and future application of diagnoses, desensitizing treatments and immunotherapies for allergic disease.

碩士
國立臺灣大學
生物化學暨分子生物學研究所
93
BG60 is an important allergen of Bermuda grass (Cynodon dactylon) pollen, which causes allergic diseases such as asthma, hey fever and allergic rhinitis. According to previous reports, BG60 has been identified as an N-linked glycoprotein and suggested that its carbohydrate moiety may be relevant to allergic diseases. Furthermore, the major structures of carbohydrate moiety were characterized as a paucimannosidic-type. In particular, a 24-residue internal peptide sequence was determined by N-terminal protein sequencing, which showed similarity to a conserved flavin-binding motif. Spectroscopic analysis also indicated BG60 was a flavoprotein. The aim of this study is to analyze these post-translational modification sites of BG60 including glycosylation and flavin-binding site. The analysis of N-linked glycosylation sites was performed by comparing the profile of MALDI-TOF MS spectra of protease-digested (trypsin and lys-C, respectively) peptide mixture with and without PNGase A treatment. The sequence of deglycosylated peptides were confirmed by MALDI MS/MS analysis. Four N-linked glycosylation sites were identified in BG60 : Asn88, Asn325, Asn354, Asn477. Furthermore, the four N-linked glycopeptides were purified by reverse-phase HPLC. The glycosylation pattern of each site was determined by MALDI-TOF MS. Our results may provide an insight into the microheterogeneity of glycoproteins. In order to identify the putative flavin-binding site, purified BG60 was limiting digested with trypsin and separated by SDS-PAGE, only the protein band which showed yellow-green fluorescence under UV illumination was subjected to in-gel tryptic digestion. The fluorescent peptide was purified using reverse-phase HPLC by monitoring of excitation at 450 nm and emission at 520 nm; which is the characterization of flavin. N-terminal protein sequence analysis of the fluorescent peptide indicated that the fluorescent compound is linked to His113. However, it was not proved by the result of MALDI-TOF MS and MS/MS analysis that the fluorescent compound purified from BG60 is a flavin. It was suggested that an unknown compound covalently linked to BG60 such as flavin derivatives that might result in similar fluorescent properties as flavin. By combination of enzymatic digestion, HPLC separation and mass spectrometric analysis, we have identified four N-linked glycosylation sites and an unknown fluorescent compound binding site. This strategy may provide an efficient and useful tool for the analysis of post-translational modification sites of protein.

碩士
國立陽明大學
臨床醫學研究所
98
Bermuda grass pollen (BGP) is an important aeroallergen in subtropical areas which can induce seasonal allergic rhinitis, conjunctivitis and asthma. In Taiwan, the positive rate of immediate skin tests to the crude extract of BGP was about 27% in asthmatic patients. Cyn d 1, the most abundant protein in BGP extracts, has been recognized as the major and group 1 allergen of BGP. It comprises 15-20% of the crude BGP extract. Cyn d 1 is a 32 to 34 kDa glycoprotein that gives a more than 76% positive IgE reaction rate in sera from BGP allergic patients. Clinically, specific immunotherapy with grass pollen extracts has been shown to be efficacious and carried out for a century. It has been shown to enhance specific IgG4 antibodies that compete with IgE antibodies to bind to allergens and thus prevents mast cell activation and histamine release. Nevertheless, since crude pollen extracts used for immunotherapy compose IgE-binding epitopes, potential risks of severe anaphylaxis can not be avoided during conventional immunotherapy. In order to prevent anaphylactic reactions, an ideal vaccine for immunotherapy should preserve human IgG4 antibody-binding epitopes but ablate IgE antibody-binding epitopes. To develop a better vaccine for immunotherapy, it is crucial to have better understanding about the IgE- and IgG4-binding epitopes. In this study, the dot immunoblotting method was applied to map human IgE- and IgG4-binding epitopes on Cyn d 1 and to further determine the essential amino acids in each epitope. In addition, mouse and rabbit specific IgG-binding epitopes on Cyn d 1 were also investigated and compared with human’s. (1) Mapping of human IgE- and IgG4-binding epitopes Synthetic decapeptides (overlapping by 5 residues) spanning the full length of the Cyn d 1 molecule were reacted with sera from 22 BGP allergic patients to locate the potential binding regions of specific human IgE and IgG4 antibodies. Decapeptides (overlapping by 7 or 9 residues) around preliminary positive regions were further synthesized to determine the exact amino acid sequences of IgE- and IgG4-binding epitopes on Cyn d 1. Using dot immunoblotting, 4 major IgE-binding epitopes on amino acids 70-79, 101-110, 159-167 and 172-181, and 3 major IgG4-binding epitopes (amino acids 70-79, 144-153, and 192-200) were identified. Among them, 3 epitopes (amino acids 70-79, 159-167, and 172-181) were recognized by both specific IgE and IgG4 antibodies. Potential epitopes specific for human IgG1, IgG2 and IgG3 antibodies were also elucidated. Their binding affinity to synthetic decapeptides of Cyn d 1 was not totally identical among 22 allergic patients. However, most of them also had strong binding activities to IgG4-binding epitopes. (2) Essential amino acids in IgE- and IgG4-binding epitopes The amino acids critical for IgE and IgG4 epitopes on Cyn d 1 were determined using synthetic peptides with single amino acid substitution by alanine. Some replacement did reduce human antibody-binding capability dramatically. Interestingly, essential amino acids between common IgE- and IgG4-binding epitopes were not identical. Future creation of hypoallergenic variants would be useful to develop safer immunotherapy. (3) Antibody-binding activities among isoforms of Cyn d 1 Among eleven isoforms of BGP, two common epitopes for IgE and IgG4 antibodies (amino acids 70-79 and 172-181) were found to possess a strong positive isoform respectively. They should be included in the test panel of BGP to increase sensitivity. (4) Mouse and rabbit IgG-binding epitopes on Cyn d 1 Both mice and rabbits were intraperitoneally immunized biweekly with 10 μg Cyn d 1 adsorbed onto 4 mg alum twice. They were then boosted monthly with10 μg Cyn d 1/alum subcutaneously for 3 times. One week after the last booster, they were bled and their sera were stored at -20℃. Epitopes recognized by mouse IgG1 and rabbit IgG antibodies were quite different. Moreover, they are also different from those of humans. Therefore, they cannot be simply recognized as human epitopes, even with some identical sites. (5) Protein structure homology modeling As the crystal structure of Cyn d 1 has not been identified, an automated protein homology-modeling server (SWISS-MODEL) was applied to extrapolate the simulated structure of the Cyn d 1 molecule and locate the epitopes. All of them have some parts of structure on the surface of the simulated protein so that they could be readily recognized by specific antibodies. In conclusion, this is the first study to map the sequential epitopes of human IgE and IgG4 on Cyn d 1. It will be helpful for the better diagnosis of BGP allergy and future development of safer and more effective immunotherapy.

博士
國立陽明大學
生化暨分子生物研究所
100
Cyn d 4, a highly basic glycosylated isoallergens from Bermuda grass pollen, shares ~70% sequence identity with the Pooideae group 4 allergens. The crystal structure of the native Cyn d 4 at 2.15-Å resolution is reported. A conserved N-terminal segment, presence only in the large isoallergens, forms extensive interactions with surrounding residues and hence greatly enhances the protein’s structural stability. Cyn d 4 contains a FAD cofactor linked to His88 and Cys152. To date all the identified bicovalent flavoproteins are oxidases and their substrates are either sugars or secondary metabolites. A deep large hydrophobic substrate-binding cleft is present. Then Cyn d 4 may be an oxidase involved in biosynthesis of a pollen-specific metabolite. The protein surface displays an unusually large number of positively charged clusters, reflecting of the high pI of ~10. Thirty-eight decapeptides covering the solvent accessible sequences did not show any significant IgE binding activity using sera with high Cyn d 4 reactivity from four patients, which suggests that the IgE epitopes of Cyn d 4 are predominantly conformational in nature. Together with several group 4 modelled structures, we suggest putative shared and unique IgE epitopes.