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.     

Mechanism of action of rat liver DNA methylase. I. Interaction with double-stranded methyl-acceptor DNA

Rat liver DNA methylase forms two types of complexes with DNA which are distinguishable on the basis of their sensitivity to ionic strength. A weak complex which is dissociable by 0.2 m-NaCl is formed at 0 °C. A more tightly bound complex which is stable to 0.2 m-NaCl is formed at higher temperatures. On the basis of a comparison of the effects of salt upon tight complex formation and upon enzyme activity, it would appear that formation of the tightly bound complex is required for DNA methylation to occur. With tightly bound enzyme the methylation of high molecular weight DNA is nearly linear for 30 minutes in the presence of salt. Under the same conditions low molecular weight DNA's give non-linear kinetics which demonstrate a sharp reduction in the rate of methylation with time. The lower the molecular weight of the DNA fragments, the more marked is the deviation from linearity. On the basis of these data it is suggested that rat liver DNA cytosine-methylase which is tightly bound to DNA is able to accomplish several methyl group transfers without becoming detached from the DNA and that the enzyme must therefore walk along the DNA helix. We calculate that the walk is probably not random, but linear, and that the rate of walk may be about 1.5 to 3.5 base pairs per second.     

The effects of infusions of ring-A-reduced derivatives of aldosterone on the antinatriuretic and kaliuretic actions of aldosterone

Infusion of Ring-A-reduced metabolites of aldosterone in adrenalectomized male rats for 4 days revealed that 5 alpha-Ring-A-reduced derivatives, 5 alpha-dihydroaldosterone (5 alpha-DHAldo; 2.5-5.0 micrograms/day), 3 alpha,5 alpha-tetrahydroaldosterone (3 alpha,5 alpha-THAldo; 5-25 micrograms/day), and 3 beta,5 alpha-THAldo (50-175 micrograms/day) possessed intrinsic Na+-retaining activity. The same infusions of 5 alpha-DHAldo, 3 alpha,5 alpha-THAldo, and 3 beta,5 alpha-THAldo, also lowered the urinary excretion of potassium. The 5 beta-Ring-A-reduced derivative 3 alpha,5 beta-THAldo did not demonstrate either of these biological properties. In another set of experiments, on the fourth day of infusion, aldosterone (0.1 microgram/rat) was administered acutely subcutaneously; none of the Ring-A-reduced derivatives altered the Na+-retaining activity of aldosterone. However, in a dose-dependent manner, both 3 alpha,5 alpha-THAldo and 3 beta,5 alpha-THAldo blunted the urinary K+-secretory effect of aldosterone; low dosages of 5 alpha-DHAldo and larger dosages of 3 alpha,5 beta-THAldo did not. Thus, the 5 alpha-reduced derivatives of aldosterone not only lowered urinary Na+ and K+ excretion in their own right, but two of them blunted the kaliuretic response of the parent mineralocorticoid, aldosterone. Further experiments will be required to determine whether these aldosterone metabolites are further metabolized or interconverted during the expression of the regulatory properties described here and whether these properties are physiologically relevant.     

Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in eukaryotes

Previously mutations in a putative protein O -mannosyltransferase (SCO3154, Pmt) and a polyprenol phosphate mannose synthase (SCO1423, Ppm1) were found to cause resistance to phage, φC31, in the antibiotic producing bacteria Streptomyces coelicolor A3(2). It was proposed that these two enzymes were part of a protein O-glycosylation pathway that was necessary for synthesis of the phage receptor. Here we provide the evidence that Pmt and Ppm1 are indeed both required for protein O-glycosylation. The phosphate binding protein PstS was found to be glycosylated with a trihexose in the S. coelicolor parent strain, J1929, but not in the pmt ⁻ derivative, DT1025. Ppm1 was necessary for the transfer of mannose to endogenous polyprenol phosphate in membrane preparations of S. coelicolor . A mutation in ppm1 that conferred an E218V substitution in Ppm1 abolished mannose transfer and glycosylation of PstS. Mass spectrometry analysis of extracted lipids showed the presence of a glycosylated polyprenol phosphate (PP) containing nine repeated isoprenyl units (C₄₅-PP). S. coelicolor membranes were also able to catalyse the transfer of mannose to peptides derived from PstS, indicating that these could be targets for Pmt in vivo .     

Differences in phosphatase modulation of alpha4beta1 and alpha5beta1 integrin-mediated adhesion and migration of B16F1 cells.

It is well established that a biphasic relationship exists between the adhesive strength of beta1 integrins and their ability to mediate cell movement. Thus, cell movement increases progressively with adhesive strength, but beyond a certain point of optimal interaction, cell movement is reduced with further increases in adhesive function. The interplay between the various kinase and phosphatase activities provides the balance in beta1 integrin-mediated cell adhesion and migration. In the present study, the significance of protein tyrosine phosphatases (PTP) and ser/thr protein phosphatases (PP) in alpha4beta1 and alpha5beta1 integrin-mediated mouse melanoma B16F1 cell anchorage and migration on fibronectin was characterized using phosphatase inhibitors. At low fibronectin concentration, alpha5beta1 functioned as the predominant receptor for cell movement; a role for alpha4beta1 in B16F1 cell migration increased progressively with fibronectin concentration. Treatment of B16F1 cells with PTP inhibitors, sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), or PP-1/2A inhibitor, okadaic acid (OA), abolished cell movement. Inhibition of cell movement by PAO and OA was associated by a reduction in the adhesive strength of alpha4beta1 and alpha5beta1. In contrast, treatment of B16F1 cells with Na3VO4 resulted in selective stimulation of the adhesive function of alpha5beta1, but not alpha4beta1. Therefore, our results demonstrate that (i) both PTP and PP-1/2A have roles in cell movement, (ii) modulation of cell movement by PTP and PP-1/2A may involve either a stimulation or reduction of beta1 integrin adhesive strength, and (iii) distinct phosphatase-mediated signaling pathways for differential regulation of the various beta1 integrins exist.     

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.