Outdoor pollen is a trigger of child and adolescent asthma emergency department presentations: A systematic review and meta-analysis.

BACKGROUND: In the context of increased asthma exacerbations associated with climatic changes such as thunderstorm asthma, interest in establishing the link between pollen exposure and asthma hospital admissions has intensified. Here, we systematically reviewed and performed a meta-analysis of studies on pollen and emergency department (ED) attendance. METHODS: A search for studies with appropriate search strategy in MEDLINE, EMBASE, Web of Science and CINAHL was conducted. Each study was assessed for quality and risk of bias. The available evidence was summarized both qualitatively and meta-analysed using random-effects models when moderate heterogeneity was observed. RESULTS: Fourteen studies were included. The pollen taxa investigated differed between studies, allowing meta-analysis only of the effect of grass pollen. A statistically significant increase in the percentage change in the mean number of asthma ED presentations (MPC) (pooled results from 3 studies) was observed for an increase in 10 grass pollen grains per cubic metre of exposure 1.88% (95% CI = 0.94%, 2.82%). Time series studies showed positive correlations between pollen concentrations and ED presentations. Age-stratified studies found strongest associations in children aged 5-17 years old. CONCLUSION: Exposure to ambient grass pollen is an important trigger for childhood asthma exacerbations requiring ED attendance. As pollen exposure is increasingly a problem especially in relation to thunderstorm asthma, studies with uniform measures of pollen and similar analytical methods are necessary to fully understand its impact on human health.

The role of seasonal grass pollen on childhood asthma emergency department presentations.

BACKGROUND: Few studies have focused on the role of grass pollen on asthma emergency department (ED) presentations among children. None have examined whether a dose-response effect exists between grass pollen levels and these asthma exacerbations. OBJECTIVES: To examine the association between increasing ambient levels of grass pollen and asthma ED presentations in children. To determine whether these associations are seen only after a thunderstorm, or whether grass pollen levels have a consistent influence on childhood asthma ED visits during the season. METHODS: A short time series ecological study was conducted for asthma presentations to ED among children in Melbourne, Victoria, and grass pollen, meteorological and air quality measurements recorded during the selected 2003 period. A semi-parametric Poisson regression model was used to examine dose-response associations between daily grass pollen levels and mean daily ED attendance for asthma. RESULTS: A smoothed plot suggested a dose-response association. As ambient grass pollen increased to about 19 grains/m(3) , the same day risk of childhood ED presentations also increased linearly (P < 0.001). Grass pollen levels were also associated with an increased risk in asthma ED presentations on the following day (lag 1, P < 0.001). CONCLUSION: This is the first study to establish a clear relationship between increased risk of childhood asthma ED attendance and levels of ambient grass pollen below 20 grains/m(3) , independent of any impact of thunderstorm-associated asthma. These findings have important implications for patient care, such as asthma management programs that notify the general public regarding periods of high grass pollen exposure, as well as defining the timing of initiation of pollen immunotherapy.

Do levels of airborne grass pollen influence asthma hospital admissions?

BACKGROUND: The effects of environmental factors and ambient concentrations of grass pollen on allergic asthma are yet to be established. OBJECTIVE: We sought to estimate the independent effects of grass pollen concentrations in the air over Melbourne on asthma hospital admissions for the 1992-1993 pollen season. METHODS: Daily grass pollen concentrations were monitored over a 24-h period at three stations in Melbourne. The outcome variable was defined as all-age asthma hospital admissions with ICD9-493 codes. The ambient air pollutants were average daily measures of ozone, nitrogen dioxide and sulphur dioxide, and the airborne particle index representing fine particulate pollution. Semi-parametric Poisson regression models were used to estimate these effects, adjusted for air temperature, humidity, wind speed, rainfall, day-of-the-week effects and seasonal variation. RESULTS: Grass pollen was a strong independent non-linear predictor of asthma hospital admissions in a multi-pollutant model (P=0.01). Our data suggest that grass pollen had an increasing effect on asthma hospital admissions up to a threshold of 30 grains/m3, and that the effect remains stable thereafter. CONCLUSION: Our findings suggest that grass pollen levels influence asthma hospital admissions. High grass pollen days, currently defined as more than 50 grains/m3, are days when most sensitive individuals will experience allergic symptoms. However, some asthmatic patients may be at a significant risk even when airborne grass pollen levels are below this level. Patients with pollen allergies and asthma would be advised to take additional preventive medication at lower ambient concentrations.

Genetic analysis of Nicotiana pollen-part mutants is consistent with the presence of an S-ribonuclease inhibitor at the S locus.

Self-incompatibility (SI) is a genetic mechanism that restricts inbreeding in flowering plants. In the nightshade family (Solanaceae) SI is controlled by a single multiallelic S locus. Pollen rejection in this system requires the interaction of two S locus products: a stylar (S)-RNase and its pollen counterpart (pollen S). pollen S has not yet been cloned. Our understanding of how this gene functions comes from studies of plants with mutations that affect the pollen but not the stylar SI response (pollen-part mutations). These mutations are frequently associated with duplicated S alleles, but the absence of an obvious additional allele in some plants suggests pollen S can also be deleted. We studied Nicotiana alata plants with an additional S allele and show that duplication causes a pollen-part mutation in several different genetic backgrounds. Inheritance of the duplication was consistent with a competitive interaction model in which any two nonmatching S alleles cause a breakdown of SI when present in the same pollen grain. We also examined plants with presumed deletions of pollen S and found that they instead have duplications that included pollen S but not the S-RNase gene. This finding is consistent with a bipartite structure for the S locus. The absence of pollen S deletions in this study and perhaps other studies suggests that pollen S might be required for pollen viability, possibly because its product acts as an S-RNase inhibitor.

The 1.55 A resolution structure of Nicotiana alata S(F11)-RNase associated with gametophytic self-incompatibility.

The crystal structure of Nicotiana alata (ornamental tobacco) S(F11)-RNase, an S-allelic glycoprotein associated with gametophytic self-incompatibility, was determined by X-ray diffraction at 1.55 A resolution. The protein has a tertiary structure typical of members of the RNase T(2) family as it consists of a variant of the (alpha+beta) fold and has eight helices and seven strands. A heptasaccharide moiety is also present, and amino acid residues that serve as the catalytic acid and base can be assigned to His32 and His91, respectively. Two "hypervariable" regions, known as HVa and HVb, are the proposed sites of S-allele discrimination during the self-incompatibility reaction, and in the S(F11)-RNase these are well separated from the active site. HVa and HVb are composed of a long, positively charged loop followed by a part of an alpha-helix and short, negatively charged alpha-helix, respectively. The S(F11)-RNase structure shows both regions are readily accessible to the solvent and hence could participate in the process of self/non-self discrimination between the S-RNase and an unknown pollen S-gene product(s) upon pollination.

Pollen tubes of Nicotiana alata express two genes from different beta-glucan synthase families.

The walls deposited by growing pollen tubes contain two types of beta-glucan, the (1,3)-beta-glucan callose and the (1,4)-beta-glucan cellulose, as well as various alpha-linked pectic polysaccharides. Pollen tubes of Nicotiana alata Link et Otto, an ornamental tobacco, were therefore used to identify genes potentially encoding catalytic subunits of the callose synthase and cellulose synthase enzymes. Reverse transcriptase-polymerase chain reactions (RT-PCR) with pollen-tube RNA and primers designed to conserved regions of bacterial and plant cellulose synthase (CesA) genes amplified a fragment that corresponded to an abundantly expressed cellulose-synthase-like gene named NaCslD1. A fragment from a true CesA gene (NaCesA1) was also amplified, but corresponding cDNAs could not be identified in a pollen-tube library, consistent with the very low level of expression of the NaCesA1 gene. RT-PCR with pollen-tube RNA and primers designed to regions conserved between the fungal FKS genes [that encode (1,3)-beta-glucan synthases] and their presumed plant homologs (the Gsl or glucan-synthase-like genes) amplified a fragment that corresponded to an abundantly expressed gene named NaGsl1. A second Gsl gene detected by RT-PCR (NaGsl2) was expressed at low levels in immature floral organs. The structure of full-length cDNAs of NaCslD1, NaCesA1, and NaGsl1 are presented. Both NaCslD1 and NaGsl1 are predominantly expressed in the male gametophyte (developing and mature pollen and growing pollen tubes), and we propose that they encode the catalytic subunits of two beta-glucan synthases involved in pollen-tube wall synthesis. Different beta-glucans deposited in one cell type may therefore be synthesized by enzymes from different gene families.

On the origin of self-incompatibility haplotypes: transition through self-compatible intermediates.

Self-incompatibility (SI) in flowering plants entails the inhibition of fertilization by pollen that express specificities in common with the pistil. In species of the Solanaceae, Rosaceae, and Scrophulariaceae, the inhibiting factor is an extracellular ribonuclease (S-RNase) secreted by stylar tissue. A distinct but as yet unknown gene (provisionally called pollen-S) appears to determine the specific S-RNase from which a pollen tube accepts inhibition. The S-RNase gene and pollen-S segregate with the classically defined S-locus. The origin of a new specificity appears to require, at minimum, mutations in both genes. We explore the conditions under which new specificities may arise from an intermediate state of loss of self-recognition. Our evolutionary analysis of mutations that affect either pistil or pollen specificity indicates that natural selection favors mutations in pollen-S that reduce the set of pistils from which the pollen accepts inhibition and disfavors mutations in the S-RNase gene that cause the nonreciprocal acceptance of pollen specificities. We describe the range of parameters (rate of receipt of self-pollen and relative viability of inbred offspring) that permits the generation of a succession of new specificities. This evolutionary pathway begins with the partial breakdown of SI upon the appearance of a mutation in pollen-S that frees pollen from inhibition by any S-RNase presently in the population and ends with the restoration of SI by a mutation in the S-RNase gene that enables pistils to reject the new pollen type.

Characterization of Rny1, the Saccharomyces cerevisiae member of the T2 RNase family of RNases: unexpected functions for ancient enzymes?

The T(2) family of nonspecific endoribonucleases (EC ) is a widespread family of RNases found in every organism examined thus far. Most T(2) enzymes are secretory RNases and therefore are found extracellularly or in compartments of the endomembrane system that would minimize their contact with cellular RNA. Although the biological functions of various T(2) RNases have been postulated on the basis of enzyme location or gene expression patterns, the cellular roles of these enzymes are generally unknown. In the present work, we characterized Rny1, the only T(2) RNase in Saccharomyces cerevisiae. Rny1 was found to be an active, secreted RNase whose gene expression is controlled by heat shock and osmotic stress. Inactivation of RNY1 leads to unusually large cells that are temperature-sensitive for growth. These phenotypes can be complemented not only by RNY1 but also by both structurally related and unrelated secretory RNases. Additionally, the complementation depends on RNase activity. When coupled with a recent report on the effect of specific RNAs on membrane permeability [Khvorova, A., Kwak, Y-G., Tamkun, M., Majerfeld, I. & Yarus, M. (1999) Proc. Natl. Acad. Sci. USA 96, 10649-10654], our work suggests an unexpected role for Rny1 and possibly other secretory RNases. These enzymes may regulate membrane permeability or stability, a hypothesis that could present an alternative perspective for understanding their functions.

Crystallization and preliminary X-ray crystallographic analysis of S-allelic glycoprotein S(F11)-RNase from Nicotiana alata.

Nicotiana alata S(F11)-RNase is an S-glycoprotein associated with gametophytic self-incompatibility. Crystals of S(F11)-RNase have been grown at room temperature using polyethylene glycol as a precipitant. A crystal diffracted to better than 1.4 A resolution at 100 K at the SPring-8 synchrotron-radiation source, indicating that it is very suitable for high-resolution structure analysis. The crystal belongs to the space group P2(1), with unit-cell parameters a = 65.86 (11), b = 44.73 (5), c = 64.36 (7) A, beta = 90.27 (9) degrees. The asymmetric unit contains two monomers, giving a crystal volume per protein mass (V(M)) of 2.05 A(3) Da(-1) and a solvent content of 39.6% by volume. A full set of X-ray diffraction data was collected to 1.55 A resolution with a completeness of 97.4%. A heavy-atom derivative has been successfully prepared with ethylmercury thiosalicylate (EMTS) and structure analysis is in progress.

A molecular description of mutations affecting the pollen component of the Nicotiana alata S locus.

Mutations affecting the self-incompatibility response of Nicotiana alata were generated by irradiation. Mutants in the M1 generation were selected on the basis of pollen tube growth through an otherwise incompatible pistil. Twelve of the 18 M1 plants obtained from the mutagenesis screen were self-compatible. Eleven self-compatible plants had mutations affecting only the pollen function of the S locus (pollen-part mutants). The remaining self-compatible plant had a mutation affecting only the style function of the S locus (style-part mutant). Cytological examination of the pollen-part mutant plants revealed that 8 had an extra chromosome (2n + 1) and 3 did not. The pollen-part mutation in 7 M1 plants was followed in a series of crosses. DNA blot analysis using probes for S-RNase genes (encoding the style function of the S locus) indicated that the pollen-part mutation was associated with an extra S allele in 4 M1 plants. In 3 of these plants, the extra S allele was located on the additional chromosome. There was no evidence of an extra S allele in the 3 remaining M1 plants. The breakdown of self-incompatibility in plants with an extra S allele is discussed with reference to current models of the molecular basis of self-incompatibility.

Structural analysis and molecular model of a self-incompatibility RNase from wild tomato.

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

A relic S-RNase is expressed in the styles of self-compatible Nicotiana sylvestris.

We surveyed ribonuclease activity in the styles of Nicotiana spp. and found little or no activity in self-compatible species and in a self-compatible accession of a self-incompatible species. All self-incompatible species had high levels of ribonuclease activity in their style. Interestingly, one self-compatible species, N. sylvestris, had a level of stylar ribonuclease activity comparable to that of some self-incompatible Nicotiana species. A ribonuclease with biochemical properties similar to those of the self-incompatibility (S-)RNases of N. alata was purified from N. sylvestris styles. The N-terminal sequence of this protein was used to confirm the identity of a cDNA corresponding to the stylar RNase. The amino acid sequence deduced from the cDNA was related to those of the S-RNases and included the five conserved regions characteristic of these proteins. It appears that the N. sylvestris RNase may have evolved from the S-RNases and is an example of a 'relic S-RNase'. A number of features distinguish the N. sylvestris RNase from the S-RNases, and the role these may have played in the presumed loss of the self-incompatibility response during the evolution of this species are discussed.

Identification of active-site histidine residues of a self-incompatibility ribonuclease from a wild tomato.

The style component of the self-incompatibility (S) locus of the wild tomato Lycopersicon peruvianum (L.) Mill. is an allelic series of glycoproteins with ribonuclease activity (S-RNases). Treatment of the S3-RNase from L. peruvianum with iodoacetate at pH 6.1 led to a loss of RNase activity. In the presence of a competitive inhibitor, guanosine 3'-monophosphate (3'-GMP), the rate of RNase inactivation by iodoacetate was reduced significantly. Analysis of the tryptic digestion products of the iodoacetate-modified S-RNase by reversed-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry showed that histidine-32 was preferentially modified in the absence of 3'-GMP. Histidine-88 was also modified, but this occurred both in the presence and absence of 3'-GMP, suggesting that this residue is accessible when 3'-GMP is in the active site. Cysteine-150 was modified by iodoacetate in the absence of 3'-GMP and, to a lesser extent, in its presence. The results are discussed with respect to the related fungal RNase T2 family and the mechanism of S-RNase action.

Molecular characterisation of an S-like RNase of Nicotiana alata that is induced by phosphate starvation.

We characterised a cDNA encoding an S-like RNase (RNase NE) from the styles of the self-incompatible plant, Nicotiana alata. RNase NE is 86% identical to an extracellular RNase from tomato cell cultures, RNase LE. DNA hybridisation experiments indicate that there are ca. 5-6 sequences related to RNase NE in the N. alata genome and that RNase NE is not linked to the self-incompatibility (S) locus. RNase NE is expressed in the styles, petals and immature anthers but not in the vegetative tissues of N. alata plants under normal growth conditions. Under phosphate-limited conditions, RNase NE expression is induced in roots but not leaves of N. alata. A transcript hybridising to RNase NE is also induced in N. plumbaginifolia cell cultures in response to phosphate starvation. RNase NE is likely to play a role in the response of N. alata to phosphate limitation, possibly by scavenging phosphate from sources of RNA in the root environment. We also discuss the evolutionary relationships between the S- and S-like RNase genes in plants.

A retrotransposon-like sequence linked to the S-locus of Nicotiana alata is expressed in styles in response to touch.

We have identified a family of repetitive sequences in the genome of Nicotiana alata named Tna1 (Transposon of N. alata). The first element we characterised was a genomic clone for the N. alata s6-ribonuclease (S6-RNase), a gene required for self-incompatibility in this species. The DNA sequence of this element resembles the integrase domain of retrotransposons of the gypsy class and is most similar to a retrotransposon from Lilium henryi. A transcript present in N.alata styles (self-incompatibility genotype S6S6) hybridized to Tna1 and accumulated in the style following either pollination or touching. This transcript was cloned from a cDNA library and was encoded by second, partial Tna1 elements. Neither the transcribed sequence nor the original Tna1 element contain an open reading frame or is likely to be able to transpose. The second element was mapped using a population of N.alata plants segregating for alleles of the self-incompatibility locus and is closely linked to the S6-allele. The Tna1 element is present in a number of Nicotiana species and appears to have been active at least twice during the evolution of this genus.

Self-incompatibility in flowering plants.

Fertilization in flowering plants begins with a pollen grain bearing the male gametes landing on the female stigma. Several mechanisms enable the stigma to discriminate between the different types of pollen that it may receive, of which the best studied is self-incompatibility. The molecules that regulate self-incompatibility are well characterized in two plant families, the Solanaceae and Brassicaceae. This list has recently been extended to include candidates for self-incompatibility molecules from the Rosaceae, Papaveraceae and Poaceae. The information provided by the sequences of these molecules gives insight into the mechanisms and evolution of self-incompatibility in the different families of flowering plants.

The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles.

Genomic clones encoding the S2- and S6-RNases of Nicotiana alata Link and Otto, which are the allelic stylar products of the self-incompatibility (S) locus, were isolated and sequenced. Analysis of genomic DNA by pulsed-field gel electrophoresis and Southern blotting indicates the presence of only a single S-RNase gene in the N. alata genome. The sequences of the open-reading frames in the genomic and corresponding cDNA clones were identical. The organization of the genes was similar to that of other S-RNase genes from solanaceous plants. No sequence similarity was found between the DNA flanking the S2- and S6-RNase genes, despite extensive similarities between the coding regions. The DNA flanking the S6-RNase gene contained sequences that were moderately abundant in the genome. These repeat sequences are also present in other members of the Nicotianae.