Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

The appearance of IgG and IgE binding components in the medium of shake cultures of Aspergillus fumigatus has been studied. Cultures were grown in synthetic asparagine medium at 35 degrees C and flasks harvested in duplicate 1, 2, 3, 4, 7 and 14 days after inoculation. The pH of the medium dropped from its initial value of 5.5 to pH 3, and then after 4 days gradually increased up to pH 7.5 in the 14-day medium. The weight of mycelium, after an initial peak followed by a slight decline, increased as the pH of the medium increased. Components able to bind IgG and IgE from pooled ABPA sera were detected by crossed immunoelectrophoresis/self-crossed radioimmunoelectrophoresis within 24 h of growth, but maximal release of both antigens and allergens coincided with the increase in pH of the medium and was seen in the 14-day culture filtrate. Two recognised "major" antigens, Ag 7 and Ag 13, detected using the relevant monospecific antisera, were present in the culture medium after 14 days of growth and similarly for Ag 3, the major allergen, although another allergen, Ag 1, was identified in the 1-day extract. None of the culture filtrates was found to contain the "C-substance" polysaccharide.

Summary A sandwich ELISA has been developed, using an affinity purified monospecific antiserum as a capture antibody, to detect specific IgG and IgG sub‐classes to a major antigen (Ag 7) of Aspergillus fumigatus in the sera of patients with allergic bronchopulmonary aspergillosis (ABPA). Significantly elevated levels of specific IgG to Ag 7 were detected in 97% of ABPA sera tested, as compared to control sera and to sera from A. fumigatus skin‐prick test positive individuals. IgG sub‐class antibody levels to Ag 7 were also determined in a similar sandwich ELISA, but using specific monoclonal antisera instead of the polyclonal anti‐IgG. Both Ag 7 specific IgG1 and IgG4 levels were found to be significantly raised in the ABPA sera compared to controls. It is proposed that this antigen‐specific ELISA may provide a more specific diagnostic test for IgG antibody detection in sera of ABPA patients.

Using a variety of immunochemical methods, including quantitative immuno-electrophoretic techniques, combined with gel filtration and iso-electric focusing, and production of monospecific antisera for identification and affinity purification, 4 major components of Aspergillus fumigatus have now been partially characterized. Numbering of these was derived from a reference allergic bronchopulmonary aspergillosis (ABPA) self-crossed radio-immuno-electrophoresis pattern of reactivity. Two major intracellular/cytoplasmic, concanavalin A (Con A)-binding antigens, Ag 7 and Ag 13, of molecular weights 150–200 and 70 kilodaltons (kD), respectively, were confirmed to be of importance for both ABPA and aspergilloma in specific sandwich enzyme-linked immunosorbent assays. A rapidly released component, Ag 5, of molecular weight 35 kD, proved both antigenic and allergenic, with aspergilloma patients having especially high-titre IgG antibodies. The major allergenic component Ag 3, of molecular weight 24 kD by gel filtration and 18 kD by SDS-PAGE was, like Ag 5, relatively heat-labile and non-Con-A-binding. Interestingly, T cell clones have been identified which respond primarily to an 18-kD fraction.

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