Limitations on the use of dihydrorhodamine 123 for flow cytometric analysis of the neutrophil respiratory burst.

Intracellular oxidation of dihydrorhodamine 123 (DHR) to the fluorescent compound rhodamine 123 (Rho123) was used to detect the production of oxygen metabolites in activated neutrophils. Total leukocyte preparations can be used in this assay, which is a great advantage when priming of the respiratory burst is studied. We have defined the conditions that should be taken into account when priming is studied with this assay.

[Determination of spectrin in erythrocytes: an important aid in the diagnosis of hereditary spherocytosis].

Objective: Assay of spectrin in erythrocytes as a diagnostic test in hereditary spherocytosis (HS).

Design: Validation of a diagnostic test.

Setting: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service in Amsterdam, the Netherlands.

Interactions between the cytosolic components p47phox and p67phox of the human neutrophil NADPH oxidase that are not required for activation in the cell-free system.

Activation of the human NADPH oxidase requires the interaction of at least four cytosolic proteins and one membrane-bound heterodimeric protein. Src homology 3 (SH3) domains and their proline-rich counterstructures have been shown to play an important role in protein-protein interactions. Because it was found that the cytosolic oxidase components p67phox, p47phox, and p40phox reside in a complex in resting neutrophils, we studied the role of SH3 domains in their interaction by use of an overlay technique.

156Pro-->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox.

Src homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain of cytochrome b558. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M.

A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox.

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils.