Parietaria is a genus of dicotyledonous weeds of the Urticaceae family including several species and its pollen grain is one of the most important allergenic sources in the Mediterranean area. Species belonging to this genus induce IgE responses in approximately 10 million people. Identification of allergens by means of independent strategies suggest that the allergens of the two more common species, Parietaria judaica and Parietaria officinalis, show molecular weights ranging between 10 and 14 kD and that the allergens of the two extracts are highly cross-reactive. Biochemical analysis and molecular cloning allowed the isolation and immunological characterization of the two major allergens of the P. judaica pollen, Par j 1 and Par j 2. Sequence comparison suggests that the P j major allergens of P. judaica belong to the nonspecific lipid transfer protein family, and three-dimensional modeling by homology has revealed that both proteins present a very conserved structural motif composed of four α-helices. Immunological analysis has shown that Par j 1 and Par j 2 are able to bind most of the P. judaica-specific IgE and some of their IgE determinants have been mapped. Recombinant Par j 1 and Par j 2 allergens have been shown to possess immunological properties equivalent to their natural counterpart and their availability represents a fundamental tool for the diagnosis and therapy of Parietaria pollen allergy.

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Background: Par j 1 represents a major allergenic component of Parietaria judaica (Pj) pollen, since it is able to induce an immunoglobulin E (IgE) response in 95% of Pj-allergic patients. It belongs to the non-specific lipid transfer protein family, sharing with them a common three-dimensional structure. Methods: Disulphide bond variants of the recombinant Par j 1 (rPar j 1) allergen were generated by site-directed mutagenesis, and the immunological activity of rPar j 1 and its conformational mutants was compared with the use of the skin prick test (SPT). The ability to bind IgE antibodies was evaluated by Western blot, ELISA and ELISA inhibition. T cell reactivity was measured by peripheral blood mononuclear cell proliferation assay. Results: The disruption of Cys14–Cys29 and Cys30–Cys75 bridging (PjA mutant) caused the loss of the majority of specific IgE-binding activity. Additional disruption of the Cys4–Cys52 bridge (PjC mutant) and the latter Cys50–Cys91 bridge (PjD mutant) led to the abolition of IgE-binding activity. On the SPT, PjB (lacking the Cys4–Cys52 and Cys50–Cys91 bridges) was still capable of triggering a type I hypersensitive reaction in 9 out of 10 patients, and PjA in 3 out of 10 patients, while PjC and PjD did not show any SPT reactivity. All the mutants preserved their T cell reactivity. Conclusion: Recombinant hypoallergenic variants of the rPar j 1 allergen described herein may represent a useful tool for improved immunotherapy.

Two cDNA clones named P9* and P1* of 794 and 631 bp, respectively, were isolated from a Λ ZAP cDNA expression library using Parietaria judaica (Pj) pollen-specific IgE antibodies from a pool of sera (n = 23) of patients allergic to Pj. Sequence analysis showed open reading frames of 176 and 138 amino acids. Both clones contain a putative signal peptide giving two mature processed proteins named Par j 1.0102 of 14,726 D and Par j 1.0201 of 10,677 D. These proteins represent isoallergenic forms of the major Pj allergen Par j 1.0101 (clone P5) previously reported. The Par j 1.0102 shared 98% amino acid sequence homology with the P5, while the Par j 1.0201 shared 89% homology. Since P1, P5 and P9 clones were expressed in Escherichia coli, and since the three allergenic proteins shared a very high degree of sequence identity and comparable binding to the Pj-specific IgE, we decided to analyze in more detail the immunological properties of only one allergen, the recombinant Par j 1.0101. The allergenic activity determined by the histamine release assay ranged between 9 and 56%, depending on the allergic patient analyzed, while it blocked approximately 40% of all the Pj-specific IgE antibodies, as detected after ELISA and cross-absorption analysis.

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A major allergen, the Par j I, was purified to homogeneity from Parietaria judaica pollen by means of ultrafiltration dialysis, preparative polyacrylamide gel chromatography and affinity chromatography through a column of Sepharose-monoclonal antibody specific for Par j I. The homogeneity of the Par j I was assessed by one single arc of immunoprecipitation both in crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis, by one single band of radiostaining after a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose and by one single peak after a size exclusion chromatography on high-performance liquid chromatography (HPLC). The homogeneity was further supported by crossed Laurell immunoelectrophoretic analysis, in that only one arc of precipitation was magnified in CIE after addition of the purified allergen. The purified Par j I allergen was capable of interacting in vitro with 70% of the human IgE specific for a crude P. judaica extract, as determined by radioallergosorbent test inhibition. The purified Par j I was capable of inducing positive reactions in vivo in skin prick tests, and of inducing release of histamine from blood containing basophils as determined by a histamine release assay. The amino acid analysis of the Par j I showed 118 amino acid residues per monomer analyzed and, among other residues, three raethionine residues were detected. The molecular weight of the Par j I estimated by HPLC and amino acid composition was 26 kilodaltons.

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DNA polymerase beta is widely distributed in the eukariotes. So far, few examples are known in which a DNA polymerase alpha -like form alone is reported. Surprisingly, DNA polymerase beta was not detected in Drosophila embryos, while it is present in the cells of multicellular species from sponge to mammals. In view of the relevance of Drosophila as a model biological system for studying the role of the various DNA metabolism enzymes in vivo we have reinvestigated the presence of the DNA polymerase beta-like form in Drosophila adult flies. Here we report the occurrence in Drosophila melanogaster adult flies of a DNA polymerase activity that, for its NEM(1) resistance, template specificity, sensitivity to ddTTP, sedimentation coefficient and nuclear localization can be classified as a beta-like form.