Figure 1.
Synthetic procedure for AX-B.
Figure 2.
1H-NMR spectra of (A) AX-B and (B) AX recorded in D2O.
Figure 3.
1H NMR of each amoxicillin: (A) AX-B and (B) AX, in presence of butylamine in D2O after 15 min. Arrows point at signals corresponding to H5 and H6, used to monitor the reaction.
Figure 4.
Modification of HSA by AX and AX-B.
(A) HSA was incubated in the presence of the indicated concentrations of Ax or Ax-B and adduct formation was assessed by western blot and detection with an anti-AX antibody (AO3.2) or with HRP-streptavidin. Exposure times were 5 minutes for AX and one second for AX-B detection, respectively. (B) HSA was incubated with AX or AX-B at 0.5 mg/ml for 16 h at 37°C. Aliquots of the incubation mixture containing the indicated amounts of total protein were analyzed by SDS-PAGE and adducts formed were detected as above. Exposure times were 2 minutes for AX and one second for AX-B detection, respectively.
Figure 5.
Competition between AX and AX-B for modification of HSA.
(A) HSA was incubated overnight with a 9-fold molar excess of AX or AX-B in bicarbonate buffer and detection of adducts was achieved by western blot with an anti-AX antibody (AO3.2) or with HRP-streptavidin, as indicated. (B) HSA was incubated in the absence or presence of AX-B, as above, followed by an overnight incubation with AX. Binding of AX was assessed by western blot with AO3.2 antibody. (C) HSA was incubated for 2 h with 80 μM AX-B, after a 16 h pre-incubation with the indicated concentrations of AX, expressed in molar excess with respect to AX-B. Aliquots of the incubation containing 2 μg of protein were subjected to SDS-PAGE and transfer, and incorporation of AX-B was assessed by detection with HRP-streptavidin. Blots shown in every case are representative of three independent assays with similar results.
Table 1.
Clinical characteristics and skin test and drug provocation test results of the patients included in the study.
Figure 6.
Recognition of AX and AX-B by the sera of allergic patients.
Shown are RAST inhibition assays using in the solid phase AXO-PLL discs and in the fluid phase AX and AX-biotin, at the indicated concentrations. Results are from selective allergic patients to AX (A, patients 1–4) and from cross-reactive allergic patients (B, patients 5–8).
Table 2.
RAST and RAST inhibition results from patients with RAST values higher than 7% to AXO-PLL and/or BPO-PLL.
Figure 7.
Modification of serum proteins by AX-B.
Total human serum was incubated in the presence of the indicated concentrations of AX or AX-B, for 16 h, after which, 70 μg of protein were analyzed by 2D electrophoresis, as described in the experimental section. (A) and (B) AX and AX-B-modified proteins were visualized by detection with AO3.2 antibody or HRP-streptavidin, as indicated. Exposure times were 3 min and 5 seconds, respectively. (C) Coomassie staining of one gel showing the positions of the identified proteins. 1, HSA; 2, transferrin; 3, 4 heavy chains of Igs; 5, 6, light chains of Igs; 7, 8, haptoglobin 2; 9, apolipoprotein A1. (D) Total human serum was incubated with 0.1 mg/ml AX-B (0.16 mM) for 2 h after a 16 h pre-incubation with the indicated concentrations of AX, expressed in molar excess with respect to AX-B. Aliquots of the incubations containing 0.5 μg of protein were resolved on a 10% polyacrylamide gel, before transfer and detection with HRP-streptavidin. The positions of transferrin, HSA and heavy and light chains of Igs, from top to bottom, are marked by asterisks.
Figure 8.
Detection of intracellular AX-B-protein adducts.
(A) Raw264.7 murine macrophages were incubated in the absence or presence of AX for 24 h, after which, formation of AX-protein adducts was assessed by SDS-PAGE of cell lysates and detection with AO3.2 antibody. The position of two of the bands detected above background is marked by arrowheads. (B) Cells were incubated with the indicated concentrations of AX-B and protein adducts present in cell extracts were detected by SDS-PAGE, transfer and detection with HRP-streptavidin. The position of endogenous biotinylated proteins is marked by arrows and that of the major labeled proteins is indicated by asterisks. (C) Cells were incubated with biotin and the presence of biotinylated proteins was assessed as in (B). (D) Cells were incubated with the indicated concentrations of AX for 24 h, after which, AX-B was added at 0.3 mg/ml and incubation was continued for an additional 24 h. Incorporation of AX-B into proteins was assessed as above.
Figure 9.
Localization and cell-type specificity of AX-B adducts.
(A) RAW264.7 macrophages were cultured in the absence or presence of 0.5 mg/ml AX-B in serum-free medium for 24 h, after which, cells were fixed with paraformaldehyde and biotin positive structures were visualized by staining with Alexa-488-streptavidin. Nuclei were stained with DAPI. Shown is a single z section at mid cell height. DIC, differential interference contrast. Bar, 20 μM. (B) RAW264.7 cells (left panel), monocytic THP-1 (middle panel) and B lymphoma cells (right panel) were incubated with AX-B and the presence of AX-B-protein adducts was analyzed by SDS-PAGE and biotin detection with HRP-streptavidin and ECL. Typically, aliquots from total protein extracts containing 15 μg of protein were analyzed. Endogenous biotinylated polypeptides are marked by arrows and the position of the polypeptides showing higher biotin incorporation after AX-B treatment is indicated by asterisks.