Kenichi Ono

Establishment and evaluation of MRK16-magnetic cell sorting assays for detecting low expression of multidrug resistance P-glycoprotein using human leukemia cell lines and peripheral blood cells from healthy donors

Two types of magnetic cell sorting assays, termed MRK16-MACS and MRK16-MACS-FACS, have been established to detect low expression level of P-glycoprotein (P-gp) using a monoclonal antibody MRK16, which recognizes a cell surface epitope of P-gp. With K-562 and U-937 cell lines, which are known to express low levels of P-gp and hence routinely used as negative control cell lines in conventional flow cytometry, both assays gave significantly positive reactivities indicating improved specificity and sensitivity of these assays. The findings in the dilution test, where P-gp-positive cells were added to P-gp-negative cells at various ratios, demonstrated that the MRK16-MACS assay is quantitative and capable of detecting small numbers of P-gp-positive cells as few as 2.5% of the total cells tested. Furthermore, specific enrichment of P-gp-expressing cells in magnetic cell sorting assays was verified by reverse transcription-polymerase chain reaction (RT-PCR) analysis and functional assay for P-gp with Rhodamine 123. The availability of such magnetic cell sorting assays may offer an approach to quantitate low level of P-gp expression.

Antibodies against synthetic carboxy-terminal peptides distinguish H-ras and K-ras oncogene products p21

Synthetic peptides corresponding to the carboxy-terminal region of H-ras, K-ras, and N-ras oncogene product p21 proteins are used to obtain antibodies specific to each ras oncogene product. The synthetic peptides of 32 amino acids are immunogenic in rabbits without being coupled to carriers. Specific antibodies are purified by absorption of the antisera with the other peptides coupled to CH-Sepharose 4B, and antibodies reacting with all three peptides are obtained by affinity chromatography. These findings imply that antibodies specific to each peptide recognize the variable carboxy-terminal region while antibodies reacting with all three peptides recognize the constant region of the carboxy-terminal amino acid sequence of p21 proteins. The affinity-purified antibodies against H-ras and K-ras peptides are shown to react specifically with c-H-ras and v-K-ras p21 proteins expressed in E. coli and eukaryotic cells, respectively. These antibodies may be useful tools to study the functional roles of p21 carboxy-terminal domain and to detect differential expression of the family of ras oncogenes in cancerous tissues. The affinity-purified anti-N-ras peptide antibody, however, fails to react with N-ras p21 in spite of its positive reactivity with the N-ras peptide.

'Ghosts' formation and their isolation from the cellular slime mold Dictyostelium discoideum.

Abstract Conditions for isolating ghosts from the cellular slime mold Dictyostelium discoideum are described. The cells were washed with a 20 mM KCl, 2.5 mM MgCl2 solution and homogenized vigorously in 15 mM lactate buffer, pH 4.8, using a tight-fitting Dounce homogenizer. The resultant spherical ghosts were purified by the dextran-polyethylene glycol aqueous two-phase system described by Brunette & Till [1], The proportion of ghosts which are finally purified by 3rd partition in the aqueous two-phase system is 5.6% of those present in the homogenate. As shown by phase-contrast and scanning electron microscopy, the plasma membrane fractions are almost completely uncontaminated by other identifiable subcellular components. On the basis of enzyme assays the ghosts isolated showed a 9- to 11-fold enrichment of alkaline phosphatase relative to the homogenate. They are free of succinic dehydrogenase, glucose 6-phosphatase but do contain some acid phosphatase and N-acetylglucosaminidase activity. Further purification using a sucrose-density gradient removes the residual lysosomal enzyme activities.

Specific detection of the precursor of ras p21 with a mouse monoclonal anti-C-terminal peptide antibody, SARA-K1.

In an attempt to clarify the post-translational modifications of ras oncogene product p21, we have established a mouse monoclonal antibody specific for the precursor of p21. The C-terminal peptide (156-188) of K(4A)-ras oncogene product p21 (p21K(4A), termed K(4A)-peptide, was used as the immunogen. In Western blotting, monoclonal antibodies were examined for their differential reactivity between two types of p21K(4A) expressed in Escherichia coli (esh-p21K(4A)) and mammalian cell (mam-p21K(4A)). One monoclonal antibody, designated SARA-K1, reacted selectively with esh-p21K(4A). The epitope for SARA-K1 was defined on tryptic peptide (177-184), containing Cys180, of the K(4A)-peptide. Pulse-chase experiments of mam-p21K(4A) synthesis at 24 degrees C revealed that SARA-K1 precipitated a 21 kDa protein within a 7 min chase but not after a 10 min chase, indicating that SARA-K1 recognizes the precursor of mam-p21K(4A). Furthermore, in Triton X-114 partitioning experiments using mammalian cells pre-treated with Mevalotin, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, SARA-K1 precipitated [35S]methionine-labeled, [3H]mevalonic acid-unlabeled mam-p21K(4A) in the aqueous phase, but did not precipitate [3H]mevalonic acid-labeled mam-p21K(4A) in either aqueous or detergent phase. The data presented clearly show that the SARA-K1 specifically recognizes the primary translational product pro-p21K(4A).