Selective nuclear protein transport was analyzed in single living cells. Hybrid proteins consisting of short stretches of the Simian virus 40 T‐antigen and of the almost complete beta‐galactosidase moiety were generated by molecular genetic methods and injected into the cytoplasm of rodent hepatoma cells. A histochemical assay showed that all proteins containing the karyophilic signal of the T‐antigen (residues 126/127‐132) were equally well accumulated by the nucleus within 15 h after injection. Microfluorimetric measurements of nuclear transport kinetics, however, revealed large differences. Proteins containing the karyophilic signal without flanking sequences were taken up by the nucleus on a time scale of hours. The same held for a protein containing T‐antigen residues 127‐147. However, a protein containing T‐antigen residues 111‐135 was accumulated by the nucleus with a half‐time of 8‐10 min reaching an equilibrium nucleocytoplasmic concentration ratio of greater than or equal to 15. Photobleaching measurements showed that, independently of subcellular localization, the mobility of all proteins was quite large. Thus, our measurements revealed a striking effect of T‐antigen residues 111‐125 on the kinetics of nuclear transport. Residues 111‐125 do not seem to contain a second karyophilic signal. Conspicuously, however, they comprise a cluster of phosphorylation sites.
SummaryBackground Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles.Objective Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. Methods Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP TM technology. Results In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. Conclusions Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy.
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