Ultracentrifugal analysis of the junction complexes of the red cell membrane cytoskeletal network: application to hereditary spherocytosis and metabolically depleted cells

A method has been developed for the assessment of the number of spectrin dimer units associated with each actin protofilament junction, in the membrane cytoskeletal network (i.e. the degree of branching) of the red cell. Ghosts are first exposed to elevated temperature at low ionic strength to dissociate some 65% of the spectrin tetramers (that link the network junctions) into dimers, without causing their release from the actin filaments. Non-ionic detergent is then added to solubilize the membrane itself with its intrinsic proteins, so as to liberate the cytoskeletal material, and the mixture is immediately examined in the analytical ultracentrifuge. The predominant components observed are isolated junctions (20 S), free spectrin dimers and the residual undissociated cytoskeletal material, with very minor components, probably corresponding to multiple junctions, linked by spectrin tetramers. The junction boundary is homogeneous within the accuracy of measurement and is taken to correspond to a complex containing six spectrin dimers, known to predominate in situ. About 17% of the total network is liberated in this form and 12% as free spectrin dimers. In hereditary spherocytosis both the size of the junction complex (as reflected by its sedimentation coefficient) and the proportion of the complex and of free spectrin liberated are indistinguishable from normal values. We conclude that the reported deficit of spectrin in hereditary spherocytosis is not reflected by a lower degree of branching of the network, and, if the membrane area is not correspondingly reduced, this must mean that the junctions are more widely spaced and the spectrin tetramers therefore more extended. In metabolically depleted cells, in which the cytoskeletal proteins are known to be extensively dephosphorylated, there is no change in the sedimentation pattern and thus no detectable loss of spectrin from the junctions or weakening in the cohesion of the cytoskeletal network.     

Multicenter evaluation of the performance and clinical utility in longitudinal monitoring of the Bayer Immuno 1 complexed PSA assay.

We conducted a multicenter evaluation of the analytical and clinical performance of the automated Bayer Immuno 1 complexed PSA (cPSA) assay, and compared assay performance to the Bayer Immuno 1 PSA assay. We sought to determine whether measurements of cPSA could be of clinical utility in the management of patients with prostate cancer. Results of the 10-day imprecision across three evaluation sites produced total CV < 2.50% and an analytical sensitivity of 0.02 microgram/L. There was an increased trend in clinical sensitivity for prostate cancer with increasing stage of disease (71-86%). Clinical specificity for patients with benign urogenital disease was 74.8%, and for other nonprostate diseases ranged from 91.1-100%. Retrospective serial monitoring of 155 patients with prostate cancer demonstrated concordance of cPSA measurements to clinical status for 97% of the patients analyzed. Results from the clinical studies using the Bayer Immuno 1 cPSA assay were comparable to results obtained with the Bayer Immuno 1 PSA assay. The Bayer Immuno 1 cPSA assay demonstrates analytical performance and clinical effectiveness in the management of prostate cancer patients during the course of disease and therapy.