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1.
Int Arch Allergy Immunol ; 116(4): 261-8, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9693275

ABSTRACT

Exposure to the hyphomycete Alternaria alternata is recognized as an important risk factor for asthma. IgE immunoblotting has been used to catalogue the number and Mr of allergens in A. alternata extracts, with estimates ranging from 10 to 30, although few are present in nearly all extracts studied. Several A. alternata allergens have been cloned, including a subunit of the major allergen Alt a 1, ribosomal P2 phosphoprotein, aldehyde dehydrogenase and a yeast YCP4 homolog. We have cloned two sequences encoding IgE-binding fragments of allergens from an A. alternata lambda gt11 cDNA library using pooled atopic sera from A. alternata-sensitive individuals. One is homologous to a region near the C-terminus of hsp70 from Cladosporium herbarum; a near-complete isoallergen variant of A. alternata ribosomal P2 protein was also cloned. Their lacZ fusion proteins had reactivities of 5 and 14%, respectively, with individual atopic sera, indicating that the corresponding allergens are both minor. This study describes one new A. alternata allergen candidate and implicates ribosomal P2 protein as an allergen thtat is stable between independently isolated clones.


Subject(s)
Allergens/genetics , Alternaria/immunology , Allergens/immunology , Allergens/metabolism , Alternaria/genetics , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Cloning, Molecular , Immunoglobulin E/immunology , Immunoglobulin E/metabolism , Molecular Sequence Data , Sequence Analysis
2.
Int Arch Allergy Immunol ; 111(4): 385-95, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8957113

ABSTRACT

Alternaria alternata is recognized as an important source of fungal aeroallergens. Alt a 1, the major allergen of this mold, is a dimer of disulfide-linked subunits that migrate in SDS-PAGE under reducing conditions at apparent M(r)s of 14,500 and 16,000. IgE antibodies to this protein are present in the sera of >90% of A. alternata-sensitive individuals. Previous studies from this laboratory showed that the N-termini twenty amino acids of the purified subunits are nearly identical. We now report the isolation of clones from an A. alternata (strain 34-016) cDNA library constructed in lambda(gt)11, using rabbit IgG antiserum against partially purified Alt a 1. One of nineteen clones selected from screens totalling 305,000 pfu (rb51) was sequenced, and determined to harbor an insert of 660 bp. An in-frame open reading frame within the cloned insert encodes a peptide of M(r) 16,960 that bears no significant homology to known allergens or proteins. The size of the rb51 transcript was determined to be approximately 0.7 kb by Northern analysis of A. alternata total RNA. The largely hydrophobic N-terminal region of the peptide contains an alpha-helical domain and other features characteristic of membrane targeting or secretory signals. The peptide sequence downstream of this region matches previously sequenced Alt a 1 N-terminal from two independent sources at 17 of 20, and 24 of 26 positions. Recombinant Alt a 1 expressed as a secreted protein in Pichia pastoris exists as a dimer in conditioned medium, as shown by immunoblotting under nonreducing conditions. Recombinant Alt a 1, like the natural allergen in A. alternata extracts, is also reactive with serum IgE from A. alternata-sensitive individuals.


Subject(s)
Alternaria/genetics , DNA, Complementary/biosynthesis , DNA, Complementary/isolation & purification , Fungal Proteins/genetics , Gene Expression , Allergens/biosynthesis , Allergens/genetics , Alternaria/immunology , Amino Acid Sequence , Animals , Antigens, Plant , Base Sequence , Blotting, Northern , Blotting, Southern , Cloning, Molecular , DNA, Complementary/genetics , Epitopes/biosynthesis , Epitopes/genetics , Fungal Proteins/biosynthesis , Genes, Fungal , Humans , Molecular Sequence Data , Pichia/genetics , Pichia/metabolism , Polymerase Chain Reaction , Protein Structure, Secondary , Rabbits , Recombinant Proteins/metabolism
3.
Virus Res ; 44(2): 79-95, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8879138

ABSTRACT

The mouse adapted strain of influenza A/FM/1/47 virus, FM-MA, has increased virulence due to mutations in HA, M1 and at least one other, unmapped, genome segment. Genetic reassortants that differ due to the HA or M1 mutations were used to define the role of these mutations in pathogenesis. Pathological changes in lungs of infected mice were assessed by hematoxylin phloxine saffron (HPS) staining, and viral infection was measured by fluorescent antibody staining of thin sections and flow cytometry of lung parenchymal cells. HA played a role in bronchiolar pathology by increasing necrosis of bronchiolar epithelium, peribronchiolar lymphocytes, and airway obstruction. The HA mutation was shown to be responsible for a 0.2 unit decreased in the pH optimum of fusion and controlled resistance to alpha and beta inhibitors of hemagglutination. Both these changes in biology may confer a replicative advantage in bronchioles seen in the first day of infection. Thus the HA mutation may have conferred a survival advantage in the extracellular lung environment. The M1 mutation resulted in improved growth in the lung and cultured cells and was associated with increases in recruitment of macrophages, spread of infection into the alveoli of the lung and interstitial pneumonia. Sequence analysis indicated that the unmapped mutation in the control of FM-MA virulence is either the K482-->R substitution in the PB2 protein or the D538-->G substitution in the PB1 protein. One or other of these mutations results in a growth advantage in infected lung but not in cultured cells as well as a further increased recruitment and infection of macrophages in the lung. Infection with virulent strains of influenza that induced increases in macrophage recruitment caused hypothermia in the mouse.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A virus/genetics , Influenza A virus/pathogenicity , Viral Matrix Proteins/genetics , Animals , Chromosome Mapping , Flow Cytometry , Fluorescent Antibody Technique , Hemagglutination Inhibition Tests , Humans , Hydrogen-Ion Concentration , Hypothermia , Influenza A virus/growth & development , Lung/pathology , Lung/virology , Macrophages/virology , Membrane Fusion , Mutation , RNA-Dependent RNA Polymerase , Viral Proteins/genetics , Virulence
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