Xxx lukm

Several reasons prompted us to reevaluate this determination and to use a simpler, nonradioactive technique.

First, the radioiodination of Luk S-PV had altered its biological activity to some extent, and second, a very high PMN concentration was used (3 × 10 PMNs/ml).

Xxx lukm-11

The supernatant was centrifuged and washed in HEPES buffer (140 m M Na Cl, 5 m M KCl, 10 m M glucose, 0.1 m M EGTA, 10 m M HEPES, 3 m M Tris base, p H 7.3).

When only PMNs were used, they were further purified as described previously (19).

Briefly, 40 ml of a dilution of blood cells in 0.9% Na Cl (1/3, vol/vol) was layered on 12 ml of J Prep (Techgen International, Voisins le Bretonneux, France). The pellet was suspended in HEPES buffer, and the contaminating erythrocytes were removed by a 45-s hypotonic lysis and subsequent washing in HEPES buffer.

The final suspension was adjusted to 6 × 10d III-linearized and dephosphorylated shuttle plasmid p CU1 (3).

The nonspecifically bound molecules of Luk S-PV do not form pores in the presence of the F component (Luk F-PV) of leucocidin.

Luk S-PV and Hlg C share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, Hlg A, Luk E, and Luk M, do not compete with Luk S-PV for its receptor. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of Luk S-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation.

The binding of the S component (Luk S-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of Luk S-PV.

The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety.

Briefly, 5 ml of injectable perfusion solution (Plasmion; Lab.

Roger Bellon, Neuilly sur Seine, France) was added to 20 ml of a dilution of white-cell-enriched blood in 0.9% Na Cl (1/3, vol/vol) and left to sediment for 30 min.

In addition, we chose flow cytometry, which allows both the analysis of low cell concentrations and fluorescence determinations.

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