Spin probe clustering in human erythrocyte ghosts

Summary A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37°C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membranebound clusters of variable size. No low probe remains at high P/L where all I(12,3) clusters in a concentrated phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.     

A selenomethionine-containing azurin from an auxotroph of Pseudomonas aeruginosa

The production and spectroscopic properties of an L-selenomethionine-containing homolog of Pseudomonas aeruginosa azurin are described. The amino acid substitution was carried out by developing an L-methionine-dependent bacterial strain from a fully functional ATCC culture. Uptake studies monitored using L-[75Se]methionine indicated that L-selenomethionine was incorporated into the protein synthetic pathway of Pseudomonas bacteria in a manner analogous to L-methionine. Several batches of bacteria were grown, and one sample of isolated and purified selenoazurin (azurin in which methionine was substituted by selenomethionine) was found (by neutron activation analysis) to contain 5.2 +/- 0.8 seleniums/copper. Correspondingly, a residual 0.35 methionines, relative to 6.0 in the native protein, were found by amino acid analysis in this azurin sample. The redox potential and extinction coefficient of this selenoazurin were found to be 333 +/- 1 mV (pH 7.0, I = 0.22) and 5855 +/- 160 M-1 cm-1 at 626 +/- 1 nm, respectively. Visible electronic, CD, and EPR spectra are reported and Gaussian curve fitting to the former spectrum allowed assignment of the selenomethionine Se----Cu(II) transition to a band found at 18034 cm-1, based upon an observed 450 cm-1 shift to the red from the analogous band position in the native protein. The data are consistent with a relatively more covalent copper site stabilizing the reduced, Cu(I), form in the selenoprotein. A role for the methionine as a modulator of the blue copper site redox potential by metal----ligand back bonding from Cu(I) is discussed in terms of a ligand sphere which limits the valence change at copper to much less than 1 during a redox cycle.     

Reduction in headache patients' medical expenses associated with biofeedback and relaxation treatments

Comparisons are made of self-reported medical costs from a sample of headache patients who underwent various combinations of relaxation training and biofeedback training. The average costs for the 2 years prior to self-regulatory treatment were $955±480 (3 SEM) for 45 patients; for the 2 years after completing treatment the average costs were $52±28 (3 SEM) for patients. Within the limitations of the study, medical costs do seem to have been markedly reduced.This research was supported by a grant from NINCDS, NS-15235.     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Galactitol accumulation by glucose-6-phosphate deficient fibroblasts: a cellular model for resistance to the complications of diabetes mellitus

When incubated in high galactose media, fibroblasts from individuals with the severe (Mediterranean) variety of glucose-6-phosphate dehydrogenase (G6PD) deficiency accumulate significantly less galactitol than do fibroblasts from matched control subjects. The effect is not observed in fibroblasts from black subjects with the more common, and milder, A- variant of G6PD deficiency. Since aldose reductase and sorbitol dehydrogenase activities in experimental and control fibroblasts are identical, the effect is most likely due to the substantial reduction in NADPH levels in severely G6PD-deficient cells. Sorbitol does not accumulate either in control or in G6PD deficient fibroblasts incubated in high glucose medium, most likely because of the action of sorbitol dehydrogenase, and the presence of a carrier-mediated glucose transport system in the cell membrane which limits the concentration of glucose that can accumulate in these cells.     

Inhibition of Transformation of Bacillus subtilis by Heavy Metals

Mercuric ions, as well as organomercuric ions and cadmium ions, can inhibit deoxyribonucleic acid-mediated transformation in Bacillus subtilis 168 without decreasing the viability of the total population. Differences in the inhibition of transformation by mercuric ions are identifiable on a temporal and concentration dependence basis. Sensitivity to low concentrations (9.2 x 10(-8) M) appears early in the uptake of deoxyribonucleic acid before the transformed markers have become insensitive to deoxyribonuclease. Resistance to "low concentrations" of Hg(2+) is kinetically indistinguishable from the requirement for magnesium in the transformation process. This inactivation is not reversed by the mercury-binding compound glutathione. Sensitivity to mercuric ions at a higher concentration (5.52 x 10(-7) M) occurs after the donor deoxyribonucleic acid has become insensitive to deoxyribonuclease. These complex interactions between mercuric ions and the process of transformation are discussed.