Maternal beliefs and practices regarding the diet and use of herbal medicines during measles and diarrhea in rural areas.
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Attitude to Health↗
Biomedical subjects
M B Singh
Publications and source records attributed to M B Singh.
Cloning of a cDNA encoding a group-V (group-IX) allergen isoform from rye-grass pollen that demonstrates specific antigenic immunoreactivity.
We have isolated and characterized the cDNA clone, 19R, that encodes an isoform of a major rye-grass pollen allergen, Lol p V [previously referred to as Lol p 1b; Singh et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1384-1388; and Lol p IX; Suphioglu et al., Lancet 339 (1992) 569-572]. Clone 19R was isolated from a rye-grass pollen cDNA expression library using grass pollen-specific immunoglobulin E (IgE) antibodies (Ab) from an allergic serum pool. The nucleotide (nt) sequence of clone 19R potentially encodes a 33.8-kDa protein of 339 amino acids (aa). It possesses a leader peptide essentially identical to the previously characterized isoform of Lol p V (Lol p VA). This indicates a mature processed 31.3-kDa protein of 314 aa, correlating well with the size of the polypeptides revealed by Western analysis of pollen proteins using IgE Ab affinity purified from recombinant fusion protein (reFP) encoded by clone 19R as solid matrix. There is no N-glycosylation motif. The protein encoded by clone 19R, designated Lol p VB, has 66.4% identity and 80.4% similarity with Lol p VA. However, a Lol p VA-specific monoclonal Ab, FMC A7, does not recognize reFP encoded by clone 19R, indicating that Lol p VB does not share this epitope. Cross-reactivity studies using affinity purified IgE Ab showed that both isoforms share similar allergenic epitopes. Immunoblot analysis using sera from a population of 30 patients showed that 80% possess IgE Ab that recognize both Lol p V isoforms. Variation occurred in the signal intensities of IgE binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Allergens↗
Identification of canary grass (Phalaris aquatica) pollen allergens by immunoblotting: IgE and IgG antibody-binding studies.
The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen in the external environment of southern Australia. Seventeen allergenic fractions of canary grass pollen, ranging in mol. mass from 14 to 100 kDa, have been identified by immunoblotting, using IgE antibodies from sera of 24/30 grass-pollen-allergic subjects. The highest frequency of IgE binding (77%) was to a major 34-kDa fraction (tentatively designated Pha a I). This protein has been partially purified and identified as a group I allergen by immunodepletion experiments, with partially purified Lol p I (from rye-grass pollen), atopic serum, and Lol p I-specific MAb. In addition, microsequencing of the N-terminus of Pha a I showed an amino acid sequence identical to Lol p I. In a separate study, IgE binding to Western blots of Pha a I, Lol p I, and Cyn d I was investigated in 24 sera and found to occur in 19/24, 18/24, and 9/24, respectively. IgE binding to all three major allergens, and to both Pha a I and Lol p I, occurred in 8/24 sera. Our findings suggest that while the N-terminal sequence of Pha a I is identical to Lol p I, there may be specific allergenic epitopes exclusive to this allergen that are important for allergenicity in southern Australia.
Allergens↗
Peptide mapping analysis of group I allergens of grass pollens.
Group I allergens, from eight different clinically important grass pollens of the Pooideae (rye-grass, canary, Kentucky bluegrass, orchard and timothy), Chloridoideae (Bermuda grass) and Panicoideae (Johnson grass, maize) were isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and subjected to limited partial proteolysis and cyanogen bromide cleavage. The generated digests were visualised on gels by silver staining. Replicate gels were blotted and screened with Lolp I-specific mAb FMC A1 and IgE binding to an allergic human serum pool, to determine the degree of antigenic and IgE-binding similarities, respectively. The highest antigenic and IgE-binding similarities were between orchard and rye-grass pollens, both in the same tribe Poeae, and among the closely related grass genera. No peptide mapping of major grass pollen allergens has previously been undertaken. This study proves peptide mapping is a powerful method for the preliminary identification and internal amino acid microsequencing of common antigenic and IgE-binding determinants of allergens, providing information that is useful in the development of reagents for the treatment of grass-pollen-associated allergies.
Allergens↗
Two consecutive thunderstorm associated epidemics of asthma in the city of Melbourne. The possible role of rye grass pollen.
OBJECTIVE: To document the clinical impact and identify the meteorological and environmental circumstances surrounding two epidemics of asthma exacerbations associated with thunderstorms in the city of Melbourne and to find a possible aetiology for these events. DESIGN: Collection of meteorological and environmental data from the Victorian Bureau of Meteorology and the Environment Protection Authority; collection of clinical data from metropolitan emergency departments and the Victorian Ambulance Service; and study of a cohort of affected patients with asthma and a control group of asthmatics who were not affected by the storms. SETTING: Tertiary institution. PATIENTS: Twelve storm-affected patients with asthma and 16 asthmatics not affected by the storms. INTERVENTION: Administration of a questionnaire, medical interview, pulmonary function tests and skin prick tests with common allergens. MAIN OUTCOME AND RESULTS: Both epidemics caused a major increase in the number of hospital attendances and admissions because of asthma exacerbation (five to ten fold rise). These events could not be related to atmospheric pollution or specific meteorological features of the storms. Patients affected by the second storm were significantly more likely to suffer from hay fever (P less than 0.05), rye grass pollen allergy (P less than 0.05) and allergy to rainfall released rye grass starch granules (P less than 0.025). CONCLUSIONS: Late spring thunderstorms in the city of Melbourne can trigger epidemics of asthma attacks. The seasonal nature of the phenomenon and the pattern of allergic responses found in affected patients suggest a possible aetiological role for rye grass pollen.
Air Pollution↗
Mechanism of grass-pollen-induced asthma.
Many asthmatics are sensitive to rye-grass pollen, but pollen grains are too large to penetrate the lower airways. Our aim was to investigate the mechanism by which rye-grass pollen causes asthma. A major allergen of rye-grass pollen, Lol pIX, is located in intracellular starch granules within pollen grains. In-vitro tests showed that pollen grains are ruptured in rainwater by osmotic shock, each grain releasing about 700 starch granules into the environment. These granules are small enough to enter the airways (less than 3 microns in diameter). The starch granules were present in atmospheric samples taken during the pollen season, and showed a 50-fold increase in atmospheric concentration on days following rainfall. Isolated granules elicited IgE-mediated responses in asthmatic patients, and 4 patients with rainfall-associated asthma who underwent an inhalation challenge test had striking bronchial constriction after exposure to starch granules. Starch granules released from rye-grass pollen seem to be capable of causing asthma.
Allergens↗
Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.
We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.
Allergens↗
Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.
We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.
Allergens↗
Isolation and developmental expression of Bcp1, an anther-specific cDNA clone in Brassica campestris.
Differential screening of a mature Brassica campestris pollen cDNA library has identified five cDNA clones that represent transcripts expressed exclusively, or at elevated levels, in pollen. We show here that the expression of one of these, clone Bcp1, is tissue specific and temporally regulated. The gene is activated during microspore development, as detected by in situ hybridization. Expression is enhanced at the time of pollen maturation and during pollen germination. In situ hybridization has also shown that Bcp 1 is activated in the tapetal cells in early anther development and continues to be expressed until tapetal dissolution. Homologous transcripts are present in pollen of other taxa of Brassicaceae including Arabidopsis, but not in pollen of any other families tested.
Amino Acid Sequence↗
Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes.
A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies. Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.
Allergens↗
Grass pollen allergens: detection on pollen grain surface using membrane print technique.
A microtechnique is described to visualize pollen allergens by making prints from the surface of mature grains on nitrocellulose membrane. The allergen Lol p I is detected by specific monoclonal antibodies or specific IgE from sera of grass-pollen-allergic patients, visualized by the immunoperoxidase method. This technique offers the potential to study the distribution of individual allergens in aerobiology samples by bright-field microscopy, and to determine the sensitivity spectra of allergic individuals to known and unknown pollen sources.
Allergens↗
Immunodetection methods for grass pollen allergens on western blots.
A comparison is made of eight different methods to detect allergenic proteins in Western blots of rye-grass pollen extracts. Horseradish peroxidase-based enhanced chemiluminescence (ECL) provides a sensitive method for the detection of allergenic proteins. The method has been modified to use more dilute solutions of ECL substrate to reduce the background, can be applied to a standard nitrocellulose membrane, and used with Kodak X-ray film. The assays can be performed rapidly, replacing use of radiolabelled probes. Increased resolution is obtained. This makes the method suitable for detection of cDNA clones on plaque lifts, and for rapid and specific purification of proteins following immunodetection on nitrocellulose membranes.
Blotting, Western↗
Molecular biology of rye-grass pollen allergens.
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Allergens↗
Effect of age, sex and birth order on the intelligence of well-nourished and malnourished slum children.
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Age Factors↗
Immunofluorescent screening of monoclonal antibodies to surface antigens of animal and plant cells bound to polycarbonate membranes.
We have developed an immunofluorescent screening method in which animal or plant cells are immobilized without fixation on polycarbonate (Nuclepore) membranes. The technique does not involve centrifugation, and virtually no cells are lost during processing. Direct microscopic analysis of antibody binding provides an extremely sensitive and reproducible screening system for hybridoma supernatants.
Animals↗
Initiation of Postmeiotic beta-Galactosidase Synthesis during Microsporogenesis in Oilseed Rape.
The synthesis of beta-galactosidase during Brassica campestris pollen development results from the transcription of the haploid genome. A quantitative cytochemical method has been developed in which 5-bromo-4-chloro-3-indoxyl-beta-d-galactoside is used as substrate giving a blue-green final reaction product. We have recently detected oilseed rape plants which are heterozygous for the beta-galactosidase locus, in which 50% of the pollen grains produced are Gal (having enzyme activity), while the other 50% are gal (enzyme deficient). The gal pollen grains served as a built-in control during microspectrophotometric determinations of enzyme activity. The present study has identified the developmental phase at which synthesis of the enzyme commenced. Activity is absent in microsporocytes, tetrads, and at microspore release. Enzyme activity is first detected in the young microspores and, by early vacuolate period, there is an increase in the rate of enzyme activity. A second period of increased enzyme synthesis occurred prior to generative cell division, although the rate is reduced in mature pollen.
Journal Article↗
Grass pollen allergens: antigenic relationships detected using monoclonal antibodies and dot blotting immunoassay.
Monoclonal antibodies against major rye-grass pollen allergens have been used to detect cross-reactive determinants in other grass pollen extracts. Antibody binding was detected by the dot blotting immunoassay in which alkaline phosphatase-conjugated anti-mouse IgG was used as secondary antibody. Taxonomically ordered variations were found between pollen of 22 grass species representing all major natural groups. One of the antibodies, that bound to the 28 to 30Kd allergen, showed high species specificity; immunoblotting showed binding to similar polypeptides in Festuca elatior but to none of the other grasses tested. This simple assay has applications in standardizing grass pollen extracts.
Antibodies, Monoclonal↗
Quantitative cytochemistry of beta-galactosidase in normal and enzyme deficient (gal) pollen of Brassica campestris: application of the indigogenic method.
The available cytochemical methods for localization of beta-galactosidase have been evaluated using pollen grains of Brassica campestris. beta-Galactosidase-deficient pollen (gal), served as a control. Azo dye methods involving naphthyl substrates showed high and nonspecific background staining to the exine. The indigogenic method, employing 5-bromo-4-chloro-3-indoxyl beta-D-galactoside (X-gal) as the enzyme substrate, gave specific opaque-blue final reaction product, while mutant pollen grains remained colourless. Final reaction product formation was blocked by D-galactono-1,4-lactone, thus demonstrating the specificity of the enzyme reaction. Using microspectrophotometry, the absorbance of the final reaction product was found to be a linear function of incubation time and section thickness in cryostat sections up to 8 micron thick and was only slightly reduced by glutaraldehyde prefixation. The validity of the indigogenic method for quantitative analysis was confirmed by using an enzyme-containing polyacrylamide gel model system and enzyme-coupled Sepharose 4B beads. Cellular sites of enzymic activity have been determined using plastic sections: final reaction product occurred in the intine wall layer and peripheral cytoplasm.
Galactosidases↗