Figure 1.
Isolation of the cell-cycle inhibiting activity from BCF.
(A) Elution profile of BCF (black line) and cell-cycle inhibiting fractions identified by BrdU incorporation in HDF cells (grey histograms). (B) Active fractions separated on a non-reducing SDS gel. The four silver-stained bands of different molecular weights analysed by LC-MS/MS are in rectangles. (C) Stacked line representation of the relative intensities displayed by each of the bands detected in SDS-PAGE in (B). Numbers correspond to molecular masses in kDa. (D) L-asparaginase activity of cell-cycle inhibiting fractions.
Table 1.
Mass spectrometry data.
Figure 2.
Cell-cycle inhibition in HDF cells.
BrdU incorporation in HDF cells incubated with: (A) UBF and BCF treated with the L-asparaginase inhibitor 5-diazo-4-oxo-L-norvaline (DONV, 20 mM) or the GGT inhibitor acivicin (ACI, 5 mM), (B) UBF and BCF derived from different H. pylori strains, (C) different concentrations of recombinant L-asparaginase. Results are expressed as a mean ± SD from 5 independent experiments. *P≤0.05, **P≤0.01.
Table 2.
GGT and L-asparaginase activity.
Figure 3.
Cell-cycle inhibition by L-asparaginase.
BrdU incorporation in cell lines exposed to variable concentration of recombinant H. pylori CCUG 17874 (A) and E. coli L-asparaginase (B) compared to untreated control cells. Boxes: magnification of the corresponding chart for L-asparaginase activities lower than 0.5 U ml−1. The points represent means (n≥3); SD not represented for clarity. (C) IC50 (L-asparaginase concentration inducing 50% cytotoxicity in U ml−1) of H. pylori and E. coli L-asparaginase (MTT assay).
Figure 4.
Expression of asparagine synthetase in cultured cell lines.
(A) Western blot analysis of HDF, AGS, MKN28, MKN7 and MKN74 cell lysates for asparagine synthetase (AS). Actin was determined as loading control. (B) Densitometric analysis of protein levels normalized to the internal loading control. Results are expressed as a mean ± SD from 3 independent experiments *P≤0.05, **P≤0.01.
Figure 5.
Subcellular localisation of L-asparaginase and ELISA assays.
(A) Western blot performed on 100 µg of the subcellular fractions of H. pylori CCUG 17874 with an anti-asparaginase antibody. From left to right: 1: pre-lytic fraction; 2: periplasmic fraction; 3 and 4: soluble and insoluble spheroplast fractions, respectively. Molecular mass markers (kDa) are indicated. (B) Total L-asparaginase activity associated with each fraction (U). (C) Catalase, carbonic anhydrase (CA) and malonate dehydrogenase (MDH) were found in the expected H. pylori subcellular fractions: periplasm (2)[34], spheroplast (3) [35] and cytoplasm (4) [36], respectively. (D) Scatter-plot of ELISA assay results on sera from 42 H. pylori positive and 43 H. pylori negative patients. Serum samples (1∶101 dilution) were tested in parallel using commercial kit 1 and plates pre-coated with recombinant H. pylori L-asparaginase. Medians indicated as horizontal bars. 21% of the total samples were anti-L-asparaginase IgG positive (+). (E) Comparison of the results obtained with commercial kit 1 and L-asparaginase-based ELISA.