Biomedical subjects

L C Tsai

Publications and source records attributed to L C Tsai.

At least 19 recordsLinked to original sources

PubMed2005

Molecular cloning and characterization of full-length cDNAs encoding a novel high-molecular-weight Dermatophagoides pteronyssinus mite allergen, Der p 11.

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application. METHODS: The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5'-3' rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children. RESULTS: Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73-80% at concentrations of 100 microg/ml. CONCLUSIONS: This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.

Adolescent↗

PubMed2003

Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin (Blo t 11).

BACKGROUND: The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms. METHODS: Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno-affinity chromatography, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and verified by MALDI-TOF MS. Recombinant full-length Blo t 11 (rFL-Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli. Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme-linked immunosorbent assay (ELISA) and skin prick test. The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies. RESULTS: Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at approximately 66 kD. The nBlo t 11 was confirmed by immunoblot analysis and MALDI-TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11. Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL-Blo t 11, has comparable IgE reactivity with the native counterpart. CONCLUSIONS: This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11. This recombinant peptide, instead of the full-length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.

Allergens↗

PubMed2002

Both TPA and SCC-Ag levels are prognostic even in high-risk stage Ib-IIa cervical carcinoma as determined by a stratification analysis.

OBJECTIVE: To determine the prognostic values of tissue polypeptide antigen (TPA), squamous cell carcinoma antigen (SCC-Ag), and carcinoembryonic antigen (CEA) in the sera of cervical carcinoma patients, especially in those with a poor prognosis. METHODS: In this retrospective study, the preoperative serum SCC-Ag, TPA, and CEA were analyzed in 779 patients with cervical squamous cell carcinoma of stage Ib-IIa who received radical hysterectomy and pelvic lymph node dissection (RAH-PLND) between 1984 and 1994. RESULTS: Due to poor predictive value and poor correlation between serum CEA and clinico-pathological factors, CEA was abandoned in this study. Elevated TPA and SCC-Ag levels, pelvic lymph node metastasis (PLNM), lymphvascular space involvement (LVSI) and deep stromal invasion (DSI) were associated with poor survival time by univariate analysis. The correlation study showed that elevated serum TPA was significantly related to PLNM, LVSI, and DSI (p = 0.004, 0.008, and 0.021, respectively), and SCC-Ag was related to PLNM and bulky tumor size (p = 0.001 and 0.02, respectively). In the multivariate analysis, only PLNM and LVSI remained independently significant indicating poor survival. Further stratification studies by PLNM and LVSI showed that elevated TPA levels could even indicate higher recurrence rates in patients with PLNM (p = 0.045), as well as SCC-Ag in patients with LVSI (p = 0.038). CONCLUSIONS: The results suggest that both elevated TPA and SCC-Ag levels depicting poor prognosis in stage Ib-IIa cervical SCC, especially indicates a group of high-risk patients who may need more aggressive therapy.

Adult↗

PubMed2001

Cytochrome b gene for species identification of the conservation animals.

A partial DNA sequence of cytochrome b gene was used to identify the remains of endangered animals and species endemic to Taiwan. The conservation of animals species included in this study were: the formosan gem-faced civets, leopard cats, tigers, clouded leopards, lion, formosan muntjacs, formosan sika deers, formosan sambars, formosan serows, water buffalo, formosan pangolins and formosan macaques. The control species used included domestic cats, domestic dogs, domestic sheeps, domestic cattles, domestic pigs and humans. Heteroplasmy was detected in the formosan macaque, domestic pig and domestic cats. The frequencies of heteroplasmy in these animals were about 0.25% (1 in 402bp). Sequences were aligned by Pileup program of GCG computer package, and the phylogenetic tree was constructed by the neighbor-joining method. The results of sequence comparison showed that the percentage range of sequence diversity in the same species was from 0.25 to 2.74%, and that between the different species was from 5.97 to 34.83%. The results of phylogenetic analysis showed that the genetic distance between the different species was from 6.33 to 40.59. Animals of the same species, both the endangered animal species and domestic animals, were clustered together in the neighbor-joining tree. Three unknown samples of animal remains were identified by this system. The partial sequence of cytochrome b gene adopted in this study proved to be usable for animal identification.

Animals↗

PubMed2001

Crystallization and preliminary X-ray diffraction analysis of the 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes.

The truncated 1,3-1,4-beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase; E.C. 3.2.1.73) from Fibrobacter succinogenes was crystallized in four different forms by the vapour-diffusion method. Form A crystals have the largest trigonal P321 unit cell, diffracting to 3.0 A resolution with four to six molecules per asymmetric unit. Form B and C crystals belong to the same monoclinic space group P2(1), but the form B unit cell is twice as large as the unit cell of form C. Form B crystals diffract to 2.5 A resolution and contain four molecules per asymmetric unit. Form C crystals diffract to 2.1 A resolution and contain two molecules per asymmetric unit. Form D crystals have the smallest orthorhombic P2(1)2(1)2(1) unit cell, containing only one molecule per asymmetric unit, and diffract beyond 2.1 A resolution. The crystallization conditions for form B and C crystals are almost identical, except that form C crystals were grown in the presence of 2 mM Ca(2+) ions. It is likely that Ca(2+) directly binds to the glucanase, leading to unit-cell shrinkage as observed in other Bacillus glucanase crystals. A self-rotation search identified non-crystallographic twofold axes that combine with the crystallographic twofold dyads to give 222 symmetry for both form A and form B crystals, indicating that the glucanase has a tendency to pack in 222 symmetry.

Bacteria↗

PubMed2001

Sequence polymorphism of mitochondrial D-loop DNA in the Taiwanese Han population.

In order to demonstrate the sequence diversity of mitochondrial D-loop DNA in the Taiwanese Han population, we established a database of 155 unrelated individuals. For each individual, the complete 980bp DNA region from the 5' end of HVI to 3' end of HVII segment was sequenced. In these 155 sequence data, 149 different haplotypes were observed, amongst these haplotypes, 144 were unique, 4 were found in 2 individuals and 1 was found in 3 individuals. When compare to the Anderson sequence, 144 transitions, 24 transversions, 5 insertions and 5 deletions were found. Eight positions exhibited more than one polymorphic sequence, six exhibited two variants while two exhibited three variants. Over the 1024bp that was analysed, pairwise differences between the sequences were 11.35+/-3.53bp. The sequence and nucleotide diversity were 0.9994 and 0.0116, respectively. The probability of two individuals randomly matching over the entire control region was 0.007. The diversity in the mitochondrial D-loop indicates the value of this locus for casework within Taiwan.

Base Sequence↗

PubMed2001

Differential regulation of placental and germ cell alkaline phosphatases by glucocorticoid and sodium butyrate in human gastric carcinoma cell line TMK-1.

The expression and regulation of alkaline phosphatase (AP) was studied in the human gastric cancer cell line TMK-1. Biochemical analysis, reverse transcription-polymerase chain reaction, and Northern blot analysis demonstrated that the cells express placental, germ cell, and intestinal AP isozymes constitutively. Dexamethasone (Dex), a synthetic glucocorticoid, was shown to specifically induce the placental AP activity to about 10-fold and sodium butyrate (NaBu) induced germ cell AP activity to about 4-fold, respectively. In contrast, these two agents showed little effect on the level of intestinal isozymes. Dex and NaBu also differentially induced the mRNA levels of the placental and germ cell APs. Northern blot analysis of the placental AP transcript in the presence of the transcription inhibitor, 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole, revealed that the half-life of placental AP mRNA is about 27 h for both the Dex-treated and untreated cells. Nuclear run-on transcription analysis indicated an apparent increase in the rate of placental AP gene transcription in Dex-treated cells. These results indicated that the effect of Dex occurred primarily by activation of the placental AP gene transcription in the cells. In order to study the direct Dex and NaBu effect on AP gene expression, the proximal promoter regions of AP genes were fused to luciferase reporter vectors. Despite the high similarity in nucleotide sequences of these two genes, transient transfection analysis demonstrated that Dex and NaBu exerted a specific stimulation only through the respective placental and germ cell AP gene promoter. Taken together, this study indicates that the expression of PAP and GCAP isozymes have specific regulatory mechanisms that can be differentially controlled by signals including glucocorticoid and NaBu.

Alkaline Phosphatase↗

PubMed2001

Directed mutagenesis of apecific active site residues on Fibrobacter succinogenes 1,3-1,4-beta -D-glucanase significantly affects catalysis and enzyme structural stability.

The functional and structural significance of amino acid residues Met(39), Glu(56), Asp(58), Glu(60), and Gly(63) of Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu(56), Asp(58), Glu(60), and Gly(63) residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k(cat) were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase in K(m) value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms of F. succinogenes 1,3-1,4-beta-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu(56), Asp(58), and Glu(60) residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-beta-d-glucanase.

Amino Acid Sequence↗

PubMed2001

A novel nomenclature for the hypervariable short tandem repeat APOAI1.

A novel nomenclature for the hypervariable microsatellite DNA, APOAI1 locus, is proposed. The complex nature of the repeat unit in this locus results in alleles separated by a single base. Polymerase chain reaction (PCR) products amplified from this locus were separated by single-strand conformation polymorphism (SSCP) electrophoresis. All the single-stranded DNA bands on the SSCP gel were removed from the gel and a second amplification performed. Homozygous DNA fragments amplified from single-stranded DNA were sequenced. From the 100 individuals studied, 30 alleles and 73 genotypes were found. A system of nomenclature for the APOAI1 locus is provided that is logical and in line with previous models. Using the primers described, the locus can be amplified and alleles designated on the basis of size. This system of nomenclature will assist in the exchange of data between laboratories for this locus.

Alleles↗

PubMed2001

Serum specific IgE reactivity to recombinant Der f 11 in asthmatic children.

This study was designed to examine the prevalence of positive serum IgE reactivity to the recombinant group 11 Dermatophagoides farinae allergen (rDer f 11) in asthmatic children in Taiwan. Using immunoblot analysis in a preliminary study of 18 asthmatic children, 13 (72.2%) reacted positively to rDer f 11 and 16 (88.9%) showed positive reactivity to D. farinae extracts. The allergenicity of rDer f 11 was further evaluated with in vivo skin tests and in vitro IgE immunodot assays in 24 mite skin-test-positive asthmatic children. Whereas 17 (70.8%) had positive skin tests to rDer f 11, 18 (75.0%) had positive serum IgE reactivity to rDer f 11. A good coincidence (87.5%) between the immunodot assay and the skin test was confirmed in these asthmatic children. Moreover, the prevalence of serum IgE reactivity to rDer f 11 was further investigated in a large panel of 49 mite skin-test-positive asthmatic children. Again, 38 (77.6%) had positive serum IgE reactivity to rDer f 11 in immunodot assays. Taken together the positive IgE reactivity to rDer f 11 in immunodot analysis ranged from 75 to 77.6% in two groups of 73 mite skin-test-positive asthmatic children. High incidence of serum IgE antibodies specific for rDer f 11 in the present study suggests that Der f 11 is a novel major allergen of house dust mites.

Adolescent↗

PubMed2000

Lysine residues 162 and 340 are involved in the catalysis and coenzyme binding of NADP(+)-dependent malic enzyme from pigeon.

Alanine-scanning site-directed mutagenesis was carried out on all conserved lysine residues of pigeon cytosolic NADP(+)-dependent malic enzyme. Only two mutant enzymes, K162A and K340A, showed significant effect on their kinetic parameters. Both mutant enzymes have K(m) values for Mn(2+) and l-malate similar to those of wild-type. The K(m) value for NADP(+) of K162A is identical to that of wild-type. However, K162A demonstrated a 235-fold decrease in the k(cat) value (0.17 +/- 0.01 vs 40.0 +/- 1.3 s(-1)). These data suggested that the side chain of K162 is important for the enzyme catalytic reaction. We propose that the epsilon-amino group of K162 may serve as a general acid to protonate the 3-carbon of enolpyruvate after decarboxylation. The K340A mutant demonstrated no effect on the k(cat) value. However, its K(m) value for NADP(+) was increased by a factor of 65 (225.7 +/- 5.07 vs 3.49 +/- 0.05 microM). We propose that the NADP(+) specificity is determined by the electrostatic interaction between the epsilon-amino group of K340 and 2'-phosphate of NADP(+).

Amino Acid Sequence↗

PubMed2000

Rapid identification of the ABO genotypes by their single-stand conformation polymorphism.

The ABO locus on chromosome 9 contains many more alleles than are currently used routinely in forensic science. The use of single-strand conformation polymorphism (SSCP) can separate sequence polymorphisms that differ by only one base. The SSCP process used allows for both single- and double-stranded polymerase chain reaction (PCR) products to be visualized. The six ABO genotypes can be differentiated by the allele-specific B and O SSCP patterns. The double-stranded DNA produced 'hybrid' bands due to heterozygous samples and allowed sequence diversity to be detected between alleles of heterozygotes. These 'hybrid' bands are valid markers to confirm genotypes of specimens.

ABO Blood-Group System↗

PubMed2000

Protein sequence analysis and mapping of IgE and IgG epitopes of an allergenic 98-kDa Dermatophagoides farinae paramyosin, Der f 11.

BACKGROUND: A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11. METHODS: The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays. RESULTS: Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642. CONCLUSIONS: Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11.

Allergens↗

PubMed2000

Expression and regulation of alkaline phosphatases in human breast cancer MCF-7 cells.

The effect of retinoic acid and dexamethasone on alkaline phosphatase (AP) expression was investigated in human breast cancer MCF-7 cells. Cellular AP activity was induced significantly by retinoic acid or dexamethasone in a time-dependent and dose-dependent fashion. A marked synergistic induction of AP activity was observed when the cells were incubated with both agents simultaneously. Two AP isozymes, tissue-nonspecific (TNAP) and intestinal (IAP), were shown to be expressed in MCF-7 cells as confirmed by the differential rate of thermal inactivation of these isozymes and RT-PCR. Based on the two-isozyme thermal-inactivation model, the specific activities for TNAP and IAP in each sample were analyzed. TNAP activity was induced only by retinoic acid and IAP activity was induced only by dexamethasone. Whereas dexamethasone conferred no significant effect on TNAP activity, retinoic acid was shown to inhibit IAP activity by approximately 50%. Interestingly, TNAP was found to be the only isozyme activity superinduced when the cells were costimulated with retinoic acid and dexamethasone. Northern blot and RT-PCR analysis were then used to demonstrate that the steady-state TNAP mRNA level was also superinduced, which indicates that the superinduction is regulated at the transcriptional or post-transcriptional levels. In the presence of the glucocorticoid receptor antagonist RU486, the dexamethasone-mediated induction of IAP activity was blocked completely as expected. However, the ability of RU486 to antagonize the action of glucocorticoid was greatly compromised in dexamethasone-mediated superinduction of TNAP activity. Furthermore, in the presence of retinoic acid, RU486 behaved as an agonist, and conferred superinduction of TNAP gene expression in the same way as dexamethasone. Taken together, these observations suggest that the induction of IAP activity by dexamethasone and the superinduction of TNAP by dexamethasone were mediated through distinct regulatory pathways. In addition, retinoic acid plays an essential role in the superinduction of TNAP gene expression by enabling dexamethasone to exert its agonist activity, which otherwise has no effect.

Alkaline Phosphatase↗

PubMed2000

Association between aminolevulinate dehydrogenase genotype and blood lead levels in Taiwan.

This study was designed to evaluate the association between the aminolevulinate dehydrogenase (ALAD) genotype and blood lead levels in a general population environmentally exposed to lead. This study population of 660 subjects was secondarily sampled from the 3000 random samples of Taiwanese general population to study the distribution of blood lead levels in the Taiwanese population. A simple assay based on the polymerase chain reaction-restriction fragment length polymorphism technique was used to determine the genotype of the ALAD gene. This study found that most of the Taiwanese population was ALAD 1-1 (95.4%). Only 4.6% (30 subjects) of population were found to be 1-2 or 2-2. It has been hypothesized that the ALAD2 allele is associated with increased absorption of lead. This study found that individuals with ALAD2 alleles had 20% higher blood lead levels than persons with ALAD1 alleles (7.83 +/- 5.95 vs 6.51 +/- 5.03 micrograms/dL). However, the difference was not statistically significant, even after adjustment for other risk factors of environmental exposure. The result supports the previous finding that individuals with ALAD2 allele had higher blood lead levels. The small sample size and large amount of variation in our study may account for the insignificant association.

Asian People↗

PubMed2000

Significance of specific IgG subclass antibodies to house dust mites in asthmatic children.

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df), the major components of house dust, are considered to be etiologic factors of extrinsic asthma. The relationships between immunoglobulin (Ig) G subclass antibodies specific for Dp (or Df) were compared in specific IgE-positive and -negative asthmatic children. METHODS: Serum levels of IgG and IgE subclass antibodies specific for Dp and Df were studied in 52 children (age, 3-13 years; mean age, 8.4 years) with asthma using enzyme-linked immunosorbent assays. The skin prick test was also used in diagnosis of the reactivity of allergic disease. RESULTS: The levels of serum-specific IgG1 and IgG2 to Dp and Df in mite-specific IgE-(or skin-test) positive asthmatic children were significantly higher than those in mite-specific IgE- (or skin test) negative children (p < 0.01). Significant correlations between the level of the specific IgE and IgG1 (r = 0.6067; p = 0.0001) or IgG2 (r = 0.5851; p = 0.0002) to Dp, and IgG1 (r = 0.3823; p = 0.0214) or IgG2 (r = 0.5057; p = 0.0017) to Df were found. The specific IgE level and skin test reactivity showed a high correlation of greater than 96% (50/52). CONCLUSIONS: The levels of mite-specific IgG subclasses were partially compatible to specific IgE levels and skin test reactivity. We conclude that house dust mite allergy is significantly associated with specific IgG1, IgG2 and IgE responses.

Adolescent↗

PubMed1999

Identification of victims of the 1998 Taoyuan Airbus crash accident using DNA analysis.

In February 1998 a civilian aeroplane carrying 196 individuals crashed in Taiwan and killed another 6 people on the ground. Although there were dental and medical records, fingerprints, photographic evidence and personal effects to identify some of the victims, DNA analysis was required to further identify severely damaged remains. From the 202 people known to have perished in the plane crash, a total of 685 fragments of human remains were subjected to DNA analysis. The analysis was carried out using nine microsatellite loci, plus amelogenin to cluster the 685 fragments into 202 groups, accounting for all the victims. To establish genetic relatedness of the victims to other victims and living relatives, additional DNA loci were used. In this case the paternity index was increased by using HLA DQA1 plus Polymarker. The same 16 DNA loci were used to test blood samples from 201 relatives to establish parent/child and sibling relationships. With the exception of 19 victims identified by non-genetic evidence, 183 victims were successfully identified by DNA typing with relatively high values of paternity index by the direct or indirect comparison of relatives. The 202 victims were from 37 different families, ranging in size from 2 to 13 members and 74 individuals known to be unrelated to any other victim. The DNA from living relatives was used to identify one member of a family group, from which other victims of the family could be identified. ABO blood group information was further used to confirm genetic relatedness within families. A comparison of the DNA profiling results to the ABO blood group of the victims showed no discrepancies with the exception of two mutations in the FGA locus. In cases of severely damaged victims from a plane crash, DNA analysis proved to be the best choice to identify victims.

Accidents, Aviation↗

PubMed1999

Sequence polymorphism of the group 1 allergen of Bermuda grass pollen.

BACKGROUND: Cyn d 1, the major allergen of Bermuda grass pollen, consists of a number of isoforms. OBJECTIVE: To examine the extent of sequence variation of Cyn d 1 isoforms at the molecular level. METHODS: A Bermuda grass pollen lambdaZAP II cDNA expression library was immunoscreened with anti-Cyn d 1 monoclonal antibodies. The reactive clones were isolated, subcloned into Escherichia coli, and sequenced. Some of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d 1 proteins. RESULTS: Ten cDNA clones were obtained, all these clones encode the full length of Cyn d 1 protein. Their deduced mature proteins can be grouped into: the long ones with 246 amino acids, and the short ones with 244 amino acids. The last two amino acids (AG) of the long Cyn d 1 are deleted in the short Cyn d 1. The remaining amino acid sequences share more than 98% identity; a total of nine amino acid variations were observed. Two recombinant Cyn d 1 proteins (rCyn d 3-2 and rCyn d 5-4) with three amino acid substitutions showed differential IgE-binding profiles. CONCLUSION: The present study extended our understanding of the primary structure of isoforms of Cyn d 1.

Allergens↗