Grantee Research Project Results
2003 Progress Report: Induction of Plant Allergens by Environmental Agents
EPA Grant Number: R830399Title: Induction of Plant Allergens by Environmental Agents
Investigators: Goldblum, Randall M. , Postlethwait, Ed , Brooks, Edward , Midoro-Horiuti, Terumi
Current Investigators: Goldblum, Randall M. , Midoro-Horiuti, Terumi , Brooks, Edward , Postlethwait, Ed
Institution: The University of Texas at Austin
EPA Project Officer: Hahn, Intaek
Project Period: December 1, 2002 through November 30, 2005
Project Period Covered by this Report: December 1, 2002 through November 30, 2003
Project Amount: $250,000
RFA: Futures Research in Natural Sciences (2001) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Hazardous Waste/Remediation , Land and Waste Management
Objective:
The objectives of this research project are to:
- Identify up to three environmental exposures that induce the production of an allergenic protein in the mountain cedar tree. This would be done by examining the effects of exposures on the expression of the pathogenesis-related (PR) protein allergen Jun a 3 in the leaves of seedlings and by examining the effects of exposing pollen grains to gaseous pollutants and ultraviolet radiation on their expression of Jun a 3. For this objective, we used salicylic acid, harpin, ozone, and ultraviolet C (UVC). Among these, salicylic acid and UVC had a significant effect on Jun c 3 expression. Because of the availability and their common ornamental use, we used Juniperus chinensis seedlings for these experiments.
- Compare the production of Jun a 3 by trees exposed to higher and lower levels of criteria pollutants in their natural environments.
- Correlate the production of Jun a 3 in leaves with that in pollen, under field conditions.
Progress Summary:
Objective 1
Effects of Salicylic Acid Stimulation Jun c 3 mRNA. Approximately 1-year-old cedar trees were maintained in a controlled environment in the laboratory. The ability of these cedar trees to mount a stress response was demonstrated by exposing them to a 5 mM solution of salicylic acid (Sigma), a potential environmental pollutant, but also the final intracellular messenger in the pathway through which environmental stress induces PR proteins. Samples were collected before and 1, 2, and 5 days after stimulation. Total RNA was extracted from the leaves using a hot-borate method. First-strand cDNA synthesis was performed with reverse transcriptase. Real-time polymerase chain reactions (PCRs) were performed with primers for Jun c 3 and a housekeeping gene. Elongation factor 1α (EF1α) was analyzed at the same time to normalize for the quantities of mRNA in each assay and the amplification efficiency in each sample. These assays were preformed at the PCR facility of the Sealy Center for Vaccine Development at The University of Texas Medical Branch. The fluorescence intensity was monitored for Jun a 3 and EF1α. The results are expressed as the fold changes of the ratio of Jun c 3 to EF1α in Figure 1.
Figure 1. The Effect of 5 mM Salicylic Acid on the Expression of Jun c 3 mRNA Was Analyzed by Real-Time PCR. The mean values of five trees of fold changes of Jun c 3:EF1α ratio from before stimulation are shown with standard deviation.
Jun c 3 expression in the leaves increased 2,000-fold by 5 days after salicylic acid stimulation, and it increased to 800-fold in the control (water-treated) trees.
Effects of UVC Stimulation on Jun c 3 mRNA. Cedar seedlings also were exposed to UVC light wavelengths. Leaves were collected before and after exposure. In these experiments, exposure to 250 μW/cm2 of UVC for 10 minutes demonstrated a 500,000-fold increase in their Jun c 3:EF1α ratios 5 days after the irradiation, those exposed for 5 minutes demonstrated a 300,000-fold increase, and no exposure controls demonstrated a 77-fold increase (see Figure 2).
Figure 2. The Time Course of the Effect of UVC on the Expression of Jun c 3 mRNA in Five Irradiated Seedlings. Values are expressed as the fold change in the Jun c 3:EF1α, relative to preirradiation values.
Preliminary Results of Jun a 3 Protein Expression in Leaves. To analyze Jun a 3 expression at the protein level, we developed methods for extracting proteins from leaves and an immunoassay using a polyclonal antibody developed against recombinant Juniper type 3 allergen. Protein was extracted from the leaves of salicylic acid-stimulated trees and control (water-stimulated) trees. The leaves were ground with a mortar and pestle under liquid nitrogen. Acetone and 50 mM citrate buffer of pH 2.8 containing 1/10th weight of polyvinylpolypyrrolidone/leaf weight were used to extract the leaf proteins, which then were precipitated with 10 percent trichloroacetic acid. The spectrum of leaf proteins was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained with Coomassie blue. Jun a 3 was quantified using polyclonal rabbit anti-MBP-Jun a 3 with purified pollen crude extract as a positive control (see Figure 3).
Figure 3. Western Blotting Analysis of Jun a 3 in Leaves. Jun a 3 content in leaf extract was analyzed with anti-MBP-Jun a 3. 30 kD bands were seen in the day 5 and day 1 samples. 80 kD bands probably represent a rubisco protein, which is the major component of plants’ chloroplasts.
Jun a 3 Protein in Crude Extract Detection by Enzyme-Linked Immunosorbent Assay (ELISA). Based on the results shown in Figure 3, we have begun to develop a more quantitative assay for type 3 allergens in protein extracts of leaf and pollen. It is apparent that we will need to either remove the rubisco or use a different capture or detecting antibody to avoid interference from what is probably nonspecific binding. We will develop a sandwich-type ELISA, in which the plates are coated with an anti-Jun a 3 antibody. Serial dilution of crude extract will be incubated in the wells. Biotinylated anti-MBP-Jun a 3 then will be added as a detection antibody. Horseradish peroxidase-streptavidin incubation followed by tetramethylbenzidine membrane substrate (Kirkegaard and Perry Laboratories) incubation will be used for the colorimetric detection system. The plates will be read at OD450 nm. This assay is being optimized, but the results are encouraging.
Objective 2
We have collected the pollen from J. ashei near a large interstate highway (I-35) at a distance of at least 1 mile from this heavy traffic corridor. Because each tree pollinates for a few days, and the pollination largely depends on the weather, it is difficult to make multiple collections of pollen from each tree. For this reason, we also have analyzed the Jun a 3 mRNA expression in cones containing pollen.
Preliminary results from the pollen from a small number of mountain cedar trees along the side and away from the highway were performed by semi-quantitative PCR. The preliminary results indicated that the pollen near the highway had a higher ratio of Jun a 3 and EF than those away from the highway. More samples need to be collected, however, to confirm this trend.
Objective 3
We have begun to collect paired samples of leaves and pollen from the same tree to validate that leaf tissue, as used in the laboratory exposure experiments, is an appropriate predictor of the Jun a 3 expression in the pollen. These samples have not been analyzed to date. We plan on collecting additional samples during the next season, starting in December 2004. These will be analyzed by real-time PCR, but hopefully also for protein content using the ELISA described above.
Future Activities:
The data from the UVC and salicylate exposure of laboratory-grown Juniper trees has provided compelling evidence that the expression of mRNA for Jun a 3 and its homologue from J. chinensis is increased dramatically by chemical and physical stimuli. The results from the gaseous pollutant ozone are less clear from our current data. Evaluation of the longitudinal ozone data suggests that some experimental variables were not well controlled during the ozone exposures. The most likely cause of this variability was desiccation of some plants in the exposure chambers because of the high velocity of air movement in chambers designed for animal exposures. We previously noted, prior to installing individual drip irrigation for each plant, that drying of the plants induced high levels of Jun a 3 expression. Therefore, we will repeat the exposure experiments in chambers with a lower air turnover number. This will require the purchase of new plants and validation of the new exposure methods.
During the first pollen-collecting season (December to mid-February), we were able to optimize the collection of pollen (and leaves) from the same trees. Pollen was collected using portable vacuum cleaners that were modified by attaching large laboratory funnels that held filter cones on the intake side. These devices are held near a branch of a tree with open cones. After starting the vacuum, the branches are shaken gently to help release the pollen. Several branches of the same tree are sampled. Leaf samples then are collected. The trees are identified using a global positioning system device and samples labeled and timed/dated accordingly. Thus, during the second season, we were able to sample more extensively.
We will need at least one more season to get an adequate number of samples to draw any conclusions concerning the role of mobile sources of pollutants on the expression of Jun a 3. Therefore, a 1-year, no-cost extension has been requested. We will consider hiring graduate students to perform collections from the same trees on days of high and low pollutions during the next season. This can be done in e proximity to an environmental monitoring station. We also may collect pollen from trees that are stressed by low-water availability.
Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
environmental management, human health, health, physical aspects, allergens/asthma, health effects, health risk assessment, physical processes, risk assessment, airway disease, allergic airway disease, allergic response, asthma, asthma triggers, cedar trees, exposure, exposure assessment, human exposure, human health risk, plant allergens, pollen, respiratory disease, sensitive populations,, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, HUMAN HEALTH, Health Risk Assessment, Epidemiology, Risk Assessments, Allergens/Asthma, Health Effects, Biochemistry, Risk Assessment, asthma, asthma triggers, sensitive populations, pollen, exposure, airway disease, allergic airway disease, respiratory disease, plant allergens, human exposure, sensitive subjects, cedar trees, allergic response, exposure assessmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.