No abstract

The effect of the chemotatic peptide, N-formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-l,3-diazole (NBD)-phallacidin to quantitate cellular Factin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by >1 nM FMLP resulted in a dosedependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 #M cytochalasin B or by a t-BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation.

To investigate the possible role of the cytoskeleton in volume regulatory responses of human promyelocytic leukemic (HL-60) cells, we monitored and modulated the F-actin content of these cells undergoing volume regulation in anisotonic media. Initial volume changes of HL-60 cells suspended in hypertonic media followed a Van't Hoff relationship, and intracellular F-actin content during volume regulatory responses in anisotonic media changed concomitantly as an inverse function of the volume shifts. These F-actin changes were shown to be an explicit function of cell volume and not tonicity of the medium. The data fit with the idea that changes in affinity of actin-binding proteins (ABPs) for actin and/or changes in the overall effective critical concentration of actin occur during acute cell volume changes, producing shifts in the relative amounts of G- and F-actin. Treatment of HL-60 cells with dihydrocytochalasin B (DHB), which perturbs cellular actin assembly, lowered resting levels of intracellular F-actin but did not prevent volume-associated F-actin changes in anisotonic media. Despite the lowered F-actin levels, HL-60 cells in the presence of DHB still undergo normal volume regulatory responses. Thus the absolute amount of intracellular F-actin does not appear to be critical for volume regulation in HL-60 cells.

Gastrointestinal toxicity is a major cause of transplant-related morbidity and mortality, emphasizing the need for corrective strategies. The peak oral mucositis score and the duration of parenteral nutritional support are useful indices of gastrointestinal toxicity because these end points are correlated with clinically significant events, including blood infections and treatment-related mortality.